The effect of the “rod-and-frame” illusion on grip planning in a sequential object manipulation task

We investigated the effect of visual context (i.e., a visual illusion) on the planning of a sequential object manipulation task. Participants (n = 13) had to grasp a rod embedded in a “rod-and-frame” illusion and insert the rod-end into a tight hole in a pre-defined way. The grip type (defined by start posture, either pronated or supinated; and end posture, either comfortable or uncomfortable) used to grasp the rod was registered as a macroscopic variable of motor planning. Different rod orientations forced the participants to switch between grip types. As expected, most participants switched between pronated and supinated start postures, such that they ended the movement with a comfortable end posture. As it has been argued that planning is dependent on visual context information, we hypothesized that the visual illusion would affect the specific rod orientation at which participants would switch into a different grip type. This hypothesis was confirmed. More specifically, the illusion affected the critical spatial information that is used for action planning. Collectively, these findings are the first to show an effect of an illusion on motor planning in a sequential object manipulation task.
Introduction
At present, there is a lively debate about the effects of visual illusions on the planning and control of discrete grasping actions (for reviews, see Glover 2004; Carey 2001). An influential model in this respect is the perception-action model of Milner and Goodale (1995; Goodale and Milner 1992, 2004). The perception-action model posits a dissociation between two functionally and structurally different visual pathways in the brain: a ventral stream processing “vision for perception” (the “what-system”) and a dorsal stream processing “vision for action” (the “how-system”). The ventral pathway processes information used for the conscious recognition and identification of objects. The dorsal pathway, however, has the purpose to guide goal directed actions, and is therefore dependent on information about the spatial properties and coordinates of an object with respect to the actor. Since these different pathways depend on different sources of visual information (i.e., context dependent information for the ventral stream and context independent information for the dorsal stream), the perception-action model predicts that a visual illusion will affect perception, but not action, a prediction for which ample evidence exists (e.g., Aglioti et al. 1995; Haffenden and Goodale 1998; Dyde and Milner 2002).
Recently, Glover proposed the planning-control model (Glover 2002; Glover and Dixon 2001a, 2001b, 2002), in which a dissociation between visual representations that subserve planning and those that are used for on-line control of action is postulated. This model posits that representations responsible for planning entail a broad range of current visual and cognitive information about (1) spatial (e.g., size, shape, orientation) and non-spatial (e.g., function, weight, fragility) properties of the target object, (2) the overarching goal of the action, and (3) the visual context surrounding the target. This information is integrated with knowledge from past experience (Glover 2004, p. 4). Representations responsible for on-line control, however, are solely aimed at minimizing the spatial error of the movement and are focused on the spatial characteristics of the target object. Support for the planning-control model has been found in experiments investigating the effects of a visual illusion on action. As an example, Glover and Dixon (2001b) had participants grasp a bar placed in front of a grated background in such a way that an orientation illusion was induced. The bar could be grasped with an overhand grip or an underhand grip. The results showed that grip choice was affected by the visual background. Hence, these findings indicate that the selection, or planning, of a particular grip type is subject to the visual context surrounding the target. In a second experiment, using the same experimental set-up, Glover and Dixon investigated planning and control by measuring kinematic parameters during transport of the hand to the bar. Contrary to the first experiment, participants were not free in their grip choice, and had to use the same, predefined grip during the whole session. In line with the predictions of the planning-control model, hand orientation was affected by the illusion in the initial part of the movement, but this effect decreased when the hand approached the bar. Thus, initial planning, as evidenced by grip type and initial kinematic parameters of the reach, was affected by the illusion, but during transport of the arm the kinematic parameterization was corrected.
Although studies that investigated the impact of the visual context surrounding the target object on the planning and control of action are abundant (e.g, Aglioti et al. 1995; Jackson and Shaw 2000; Danckert et al. 2002; Franz 2001; Franz et al. 2005; Mendoza et al. 2006; Van Doorn et al. 2007), the evidence is restricted to simple prehension tasks that did not demand any further action with the grasped object: the goal of the action was to merely to grasp and lift the object. No studies have yet investigated the effect of visual context in situations where the overarching goal of the action does not coincide with simply grasping and lifting the object. This is surprising, since tasks such as a sequential object manipulation task, in which a target is grasped for a specific purpose, provide a method ‘par excellence’ to evaluate planning processes. By using a sequential object manipulation task, it can be assessed whether planning processes also take the visual context surrounding of an object into account when the overarching goal of the action requires further manipulation of the grasped object.
A distinctive feature of sequential object manipulation tasks is that they require anticipatory planning, i.e., the forthcoming perceptual-motor demands associated with the goal of the action sequence need to be taken into account when initially grasping an object (cf., Johnson-Frey et al. 2004). In other words, the type of grasp is not only determined by the characteristics of the target object and its visual surrounding, but must also accommodate the ensuing task requirements. Ample evidence for such anticipatory planning can be found in the studies of Rosenbaum and co-workers. They showed that the selection of a particular grip type is indicative for motor planning. Participants preferred to grasp an object with a grip type that enabled them to end the task in a comfortable posture, the so-called ‘end-state comfort’ effect (Rosenbaum and Jorgensen 1992; Rosenbaum et al. 1992, 1993, 1996; Cohen and Rosenbaum 2004; Short and Cauraugh 1999). Stated differently, participants sacrifice initial comfort for the sake of final comfort, implying anticipatory motor planning. For example, in the Rosenbaum and Jorgensen study (1992) participants had to grasp and rotate a bar that was mounted on a clock-face. Positions on the clock-face were separated by 45°, and participants were instructed to take hold of the bar and rotate it to a pre-defined target position. The results showed that participants adapted their initial posture, such that it enabled them to end the task in a comfortable posture. Obviously, to attain this comfortable end posture, they switched between overhand and underhand grip types when grasping the bar (for similar findings from a different theoretical background, see Kelso et al. 1994). Summing up, the studies of Rosenbaum et al. showed that initial grip type is indicative for motor planning in a sequential object manipulation task, and second, they showed that initial comfort is sacrificed to attain posture comfort at the end of the task. Additionally, previous studies have shown similar effects for kinematic parameterization. For example, Marteniuk et al. (1987, see also Gentilucci et al. 1997) showed that the goal of the second movement was reflected in movement kinematics of the first movement. Likewise, Steenbergen et al. (1995) showed that the strength of joint couplings of the first movement was critically dependent on the task constraints of the second movement.
At present, no study has scrutinized the effects of visual context on movement planning in a sequential task that requires anticipatory planning. Earlier studies on the effects of visual context on the planning of action used simple grasping movements. For instance, in the experiments of Glover and co-workers the action goal was to grasp the bar, no further manipulation was required. Hence, the posture with which the bar was grasped was identical to the posture at the end of the task. Consequently, no conflict occurred between comfort of the initial posture and comfort of the end posture. In a sequential task however, the initial posture with which an object is grasped and the posture at the end of the task are not necessarily the same because additional movements are made after grasping the object. Therefore, when planning the initial posture in a sequential object manipulation task, participants have to take the constraints arising from the end posture into account as well, i.e., they are engaged in anticipatory planning (Johnson-Frey et al. 2004).
In the present study, the effect of visual context on anticipatory planning of a sequential object manipulation task was investigated. Specifically, participants had to grasp a rod embedded in a “rod-and-frame” illusion (i.e., a rod surrounded by a tilted frame) and subsequently place the rod-end vertically in a tight hole. Both, rod and frame could be independently rotated. Following Rosenbaum et al. (1992) we expected adaptations in the initial posture such that a comfortable end posture is reached. Specifically, participants are expected to switch between pronated and supinated initial postures at a specific rod orientation. Based on the planning-control model (Glover 2002, 2004), in which context effects are not distinguished with respect to the different components of planning, it is hypothesized that the exact rod orientation at which this switch occurs is affected by the rotation of the frame.
Method
Participants
Thirteen right-handed college students (three males, ten females), aged 18–27 years (mean age 22.6 year/month, SD 2.10 year/month) participated in the experiment for money or course credit (see Table 1 for participant information). All participants had normal or corrected-to-normal vision, were naïve to the purpose of the experiment, and had no known neurological deficits. This study was approved by the local ethics committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
Table 1Participant informationPartM/FAge (year)PercepPro: supicomf:uncomfStrategy1.F230.90100:125225:0Comfortable ender2.F220.9224:201225:0Comfortable ender3.F250.97101:124223:2Comfortable ender4.F200.82108:117225:0Comfortable ender5.F21×52:173224:1Comfortable ender6.M250.9082:143222:3Comfortable ender7.F270.7987:138225:0Comfortable ender8.F200.90123:102225:0Comfortable ender9.M260.80146:79225:0Comfortable ender10.M27×138:87225:0Comfortable ender11.F180.90225:038:187Pronation starter12.F190.74225:0144:81Pronation starter13.F190.70224:156:169Pronation starterPart: participant number; M/F male/female; age: age in years; Percep: Perception task—percentage correct answers; Pro:supi: Start posture—number of pronated start postures: number of supinated start postures; Comf:uncomf: End posture—number of comfortable end postures: number of uncomfortable end postures; Strategy: strategy used in the action task (see text for description)
Experimental set-up and apparatus
The participants were comfortably seated in a chair positioned in front of a table upon which the experimental set-up was placed (see Fig. 1). The stimulus consisted of a white 3D “rod-and-frame” illusion that was placed in front of a black curtain (220 × 105 cm). This curtain was used to prevent any visual cues of veridical frames of reference, such as the ceiling or the floor. Both the rod (length: 15 cm, diameter: 3.5 cm) and the surrounding frame (30 × 30 × 2.3 cm) could be rotated independently such that the rod-and-frame illusion was created. The rod had a grey marker on one side signifying the end that had to be placed upwards in the hole (diameter: 5 cm) of a box. After the participant had placed the rod in the hole, an experimenter sitting next to the participant replaced the rod to the set-up and scored the used grip type. Rotation of rod and frame was performed manually by a second experimenter who sat behind the curtain. Participants wore liquid crystal occlusion goggles to prevent them seeing the rotation of the frame and rod in-between trials. The goggles could be switched from opaque to transparent in less than 30 ms.
Fig. 1Schematic drawing of the experimental setup, viewed from above (a) and a photograph of the first author grasping the rod (b)
Procedure
The study consisted of two experimental sessions that were conducted in succession. First, an action task was performed, second we performed a perception task to assess participants’ perceptual sensitivity for the illusion [these tasks are denoted as (1) Action task and (2) Perception task in what follows]. Standard rest breaks were present between sessions, and on participants’ demands.
Action task
The action task consisted of a pre-measurement and the main experiment. The procedure for both was as follows. A trial started when the participant pressed the button on the button-box with the index finger of the preferred (right) hand. Subsequently, the goggles were closed and the second experimenter manually changed the rod and frame orientation. When ready (i.e., within 2 s) the goggles opened, which was the start-signal for participants to grasp the rod as quickly as possible and place it vertically with the marker facing upwards in a hole of a tight fitting box that was located in front of them, slightly to the right of the body midline. Participants were asked to grasp the rod with a power grip, i.e., with the thumb on one side of the rod and the fingers on the other side. Once the rod was grasped, participants were not allowed to change the grip type during rotation of the rod. This was necessary, because it urged participants to plan the task prior to grasping the rod. If this had not been the case and participants were allowed to manipulate the rod in-hand, then it would not have been strictly necessary for participants to plan the movement prior to grasping the rod.
As dependent variable, the grip type that participants used to grasp the rod was measured. The grip types were evaluated on two criteria: the start posture of the hand and the end posture of the hand. The start posture was scored as either a “pronated” (overhand) or a “supinated” (underhand) posture. The end postures were scored as “comfortable” when a grip with the thumb towards the marker was used and as “uncomfortable” when a grip with the thumb away from the marker was used (see also, Steenbergen et al. 2000; Rosenbaum and Jorgensen 1992). Since the start and end posture are not fully independent of each other, the combination of start and end posture was labeled as the grip type. Three different grip types were distinguished: grip type 1, a pronated initial posture resulting in a comfortable end posture, grip type 2, a supinated initial posture resulting in a comfortable end posture and grip type 3, a pronated initial posture resulting in an uncomfortable end posture (see Fig. 2). The combination of a supinated initial posture resulting in an uncomfortable end posture was theoretically possible. However, this combination was never used, and will therefore not be mentioned in what follows.
Fig. 2The grip type scoring system used to establish the grip type that participants used. Grip types were defined by the combination of the initial posture (pronated or supinated) and the end posture (comfortable or uncomfortable). Explanation, see text
With respect to the grip type, our primary interest was the rod orientation at which a switch into another grip type occurred. The rod orientation at which there was an equal chance to observe both grip types was denoted the “switch point”.
Pre-measurement
As the location of the switch point differed between individuals, we performed a pre-measurement prior to the main experiment. In this pre-measurement the individual switch point of each participant was established. In general, switches in grip types occur in the lower half of the “clock face” (e.g., Rosenbaum et al. 1992; Steenbergen et al. 2000), but individual differences are present as to the exact orientation of the rod where the switch occurs. During the pre-measurement the frame was not rotated. Rod orientations were presented in a range of 180°, from the horizontal rod orientation with the marker on the left side (denoted as −90°), via the vertical rod orientation with the marker facing downwards (denoted as 0°) to the horizontal rod orientation with the marker on the right side (denoted as 90°). Thirteen rod orientations were tested, separated by equal angles of 15° (see Fig. 3). Every rod orientation was presented three times in a completely randomized order, resulting in a total of 39 trials. The switch point was determined by the rod orientation where participants switched between two different grip types, thus, at this rod orientation there was an equal chance to observe both grip types. For most participants the switch point was restricted to one rod orientation. When the grasping pattern consisted of a range of rod orientations, the mathematical middle of that range was taken to be the switch point for that participant. The pre-measurement took approximately 15 min.
Fig. 3Schematic drawing of the 13 rod positions used in the pre-measurement of the action task. In this figure the rod is oriented at 45°. The black side of the rod represents the marker. Note that the color coding is inconsistent with the experiment where we used a black background, whereas the rod and the surrounding frame were colored white (see Figs. 1b, 2 )
The main experiment
The rod orientations during the main experiment were normalized to the individual switch points, which allowed us to study the individual switch region into detail without overloading participants with too many trials. Measurements were performed in a range of 80° surrounding the individual switch point, separated by angles of 10°. This resulted in a total of nine rod orientations that were tested in the main experiment (−40°, −30°, −20°, −10°, 0°, 10°, 20°, 30°, 40° relative to individual switch point). Negative orientations are clockwise rod orientations compared with the individual switch point, whereas positive orientations are directed counterclockwise to the switch point. During the experiment we also manipulated the orientation angle of the frame, such that the “rod-and-frame” illusion was created. The frame was rotated in either a clockwise (CW) or a counterclockwise (CCW) direction. A total of five frame orientations were used (20° CCW, 10° CCW, 0°, 10° CW, 20° CW) yielding a total of 45 unique conditions. In each condition, five trials were performed in a completely randomized order. The main experiment, involving 225 trials, took about 45 min for each participant.
Perception task
We performed a perception task to assess participants’ perceptual sensitivity for the illusion. It was examined whether different rotations of the surrounding frame affected the perceived orientation of the rod. To that end, two rod-and-frame combinations were sequentially shown to the participant. First, a rod surrounded by a tilted frame was shown, followed by either the same or a different oriented rod surrounded by Frame 0°. In between presentations, the goggles were closed for less than 2 s. Participants had to report if the orientation of the rod was the same or different in the two displays. In the majority of the trials the rod orientation did not change between presentations (for example, when the first display was a combination of Rod −30° and Frame 20° CCW, the second display combined Rod −30° with Frame 0°). In this perception task, 4 frame rotations × 9 rod orientations × 3 repetitions were tested, yielding 108 trials. In addition, we also added 72 “catch trials” (4 Frame rotations × 9 rod orientations × 2 directions of rod changes), where the rod orientation actually did change between the two presentations, either 10° CW or 10° CCW. The main reason to add catch trials was to prevent that participants could anticipate that the two rods were the same in all trials. However, catch trials were not used in the analyses. The total of 180 trials was presented in a completely randomized order. The perception-task took about 45 min to be carried out.
Data analysis
Action task
Analysis of pilot recordings revealed that participants used two strategies to perform the action task. Although all participants used grip type 1 in some of the trials, at the individual switch point differences in grip type choice appeared. While most of the participants switched to an underhand initial posture resulting in a comfortable end posture (grip type 2), some participants switched to an overhand initial posture resulting in an uncomfortable end posture (grip type 3). Consequently, two movement strategies could be delineated. One group of participants switched between grip type 1 and grip type 2 and always ended with a comfortable end posture (this strategy is denoted as “comfortable enders”), whereas the other group of participants switched between grip type 1 and grip type 3 and always started with a pronated initial posture (this strategy is denoted as “pronation starters”).
At the individual switch point every participant used grip type 1 in approximately 50% of the trials, irrespective of the strategy employed, because participants either switched between grip type 1 and grip type 2 (comfortable enders) or between grip type 1 and grip type 3 (pronation starters). This allowed us to collapse the data and to use the same scoring method for both strategies, that is, the frequency of grip type 1. For every participant individually, logistic (S-shaped) functions were fitted through the mean frequency of grip type 1, separately for the five different frame orientations and on the basis of a least squares fitting method (see Van Doorn et al. 2007 for a similar method). The function was of the form where y is the assigned score, i.e., the location of the switch point, x is the rod orientation, c is the rod orientation of the switch point and k is a measure of the slope at that point Using this method, for every participant the location of the switch point (i.e., the rod orientation where a participant switched between grip types) was determined for the five frame orientations. In order to calculate the illusion effect, the value of the switch point in the control condition (0° frame rotation) was subtracted from the value of the switch point in the experimental conditions (where the frame was rotated). As our prime interest was the effect of visual context on planning, rather than the direction of the illusion effect, we used absolute difference scores. Moreover, the direction of the illusion effect was not similar among participants, a finding that is not uncommon in the “rod-and-frame” illusion literature (e.g., Beh and Wenderoth 1971; DiLorenz and Rock 1982). The absolute difference scores were analyzed using a repeated measures Analysis of Variance (ANOVA) with frame as within subjects factor.
Perception task
The number of errors per condition were analyzed using a 4 (frame: 20° CCW, 10° CCW, 10° CW, 20° CW) × 5 (rod: −20°, −10°, 0°, 10°, 20°) repeated measures ANOVA. Frame as a factor in the ANOVA denoted the first frame that is presented to the participant. The second frame was always the same, i.e., 0°.
Results
Action task
During the experiment, participants showed the same grasping behavior as in the pre-measurement phase, that is, they switched between different grips at a particular rod orientation. The average switch point over all conditions was at rod orientation −3° (for the “comfortable enders” at rod orientation −6° and for the “pronation starters” at rod orientation 6°), all were in the lower half of the clock face. For the negative rod orientations (i.e., rod orientations that are rotated clockwise compared with the individual switch point) participants used grip type 1, which is an overhand initial posture resulting in a comfortable end posture. Conversely, at the positive rod orientations (i.e., rod orientations that are rotated counterclockwise compared with the individual switch point) the grip patterns were less consistent. Most participants (n = 10) switched to an underhand initial posture leading to a comfortable end posture, while some (n = 3) switched to an overhand initial posture leading to an uncomfortable end posture (“comfortable enders” and “pronation starters”, respectively, see also Table 1).
To answer our main research question (does visual context affect anticipatory planning?), we analyzed the effect of frame orientation on the location of the switch point. For each participant individually, we calculated at which rod orientation they switched between grips for all frame orientations, using a logistic function. This way, we could calculate the magnitude (in degrees) by which the switch point had shifted in the experimental conditions compared with the control condition. In Fig. 4 the data of four participants are shown (participants 9, 10, 12 and 13). In the figure, the different frame orientations are depicted on the x-axis (with Frame 0° as the control condition), whereas the y-axis represents the location (i.e., rod orientation) of the switch point. It can be derived that the location of the switch point is different in the control condition and the experimental conditions. However, the effect of frame was not in the same direction for all participants. Therefore, absolute different scores between the switch point of the control condition (i.e., Frame 0°) and the switch point in the four experimental conditions (i.e., Frame 10° CW, Frame 20°, Frame 10° CCW and Frame 20° CCW) were calculated as a measure of the illusion effect. The mean absolute illusion effect (i.e., the amount of degrees that the switch point had shifted compared with the control condition) was 5.3° for Frame 20° CCW, 9.3° for Frame 10° CCW, 7.4° for Frame 10° CW and 6.0° for Frame 20° CW (see Fig. 5). A repeated measures ANOVA revealed a significant effect of Frame [F(4,48) = 3.29, P < 0.05 with Greenhouse Geisser correction for sphericity]. Pairwise comparisons showed that the illusion effect of Frame 20° CCW, Frame 10° CCW and Frame 20° CW were significantly different from the control condition (all P < 0.05).
Fig. 4Location of the switch point (i.e., rod orientation) in the five frame rotation conditions in four participants (9, 10, 12, 13). On the x-axis the five frame orientations are depicted, whereas the y-axis represents the rod orientation of the switch pointFig. 5The absolute effect of frame rotation on the switch point for the four frame orientations (averaged over all participants). Error bars indicate 2 SE of the mean. The absolute effects are calculated by subtracting the value of the switch point in the control condition from the experimental conditions, hence, the control condition is not depicted here. On the x-axis the different frame orientations are plotted, whereas the y-axis represents the magnitude (in degrees) by which the switch point was shifted compared with the control condition
Perception task
Data of two participants (participants 5 and 10, see Table 1) were not used for analyses due to technical problems. The mean score of all participants was 0.85 (SD 0.084), indicating that in 85% of the trials participants correctly reported that the perceived rod orientation in the two presentations was not different. The percentages of correct answers varied between 70% (participant 13) and 97% (participant 3). The mean score in the ‘comfortable end posture’ group was 87% compared with 78% in the “pronation start posture” group. However, this between subjects effect of strategy just failed to reach significance [F(1,9) = 3.74, P = 0.085]. A repeated measures ANOVA on the total number of errors revealed a significant effect of Frame [F(3,27) = 4.14, P < 0.05]. The percentages of correct answers were 86% for Frame 20° CCW, 90% for Frame 10° CCW, 87% for Frame 10° CW and 77% for Frame 20° CW. Post hoc comparisons showed significant differences between Frame 20° CW and Frame 10° CW (P = 0.051) and between Frame 20° CW and Frame 10° CCW (P < 0.05).
Discussion
The purpose of the work reported here was to evaluate the influence of visual context on the planning of a sequential object manipulation task. Earlier research on the effects of visual illusions on action was limited to simply grasping a target object without any further purpose. By contrast, in the present study we asked participants to grasp a target object to subsequently place it in a hole. This task requires anticipatory planning, in which constraints arising from the end posture prevail in initial grip choice. That is, the initial grip must accommodate the upcoming movements. As far as we know, no other study has scrutinized visual context effects in such a sequential, object manipulation task. In our study, a rod was embedded in a typical “rod-and-frame” illusion configuration. We used a wide range of rod orientations that would force participants to switch between different grip types if they were to reach a comfortable posture at the end of the task. The effect of visual context on anticipatory planning processes was investigated by measuring if the location of the switch point shifted when the surrounding frame was tilted.
The main finding of our study was that the frame manipulations affected the location of the switch point (i.e., the rod orientation where participants switched between grip types), and thus the motor planning of the initial grip type towards the target object. Although earlier findings have already shown that the kinematics and joint couplings in the first movement towards a target object are affected by the upcoming second movement (Gentilucci et al. 1997; Marteniuk et al. 1987; Steenbergen et al. 1995), our results extend these finding by showing that initial grip planning is also affected by the visual context. However, in line with earlier findings on the illusion effects of the “rod-and-frame illusion” (e.g., Beh and Wenderoth 1971; DiLorenz and Rock 1982) the results did not show a consistent direction of the illusion effect among participants. This phenomenon is due to the complex interaction between the specific location of the individual switch point with the frame orientation and the individual sensitivity for the illusion.
Our results are in line with at least three contemporary models that make specific predictions about the effect of illusions on action (planning). First, following the predictions of Glovers’ model (2004) we hypothesized that the visual context would affect the specific rod orientation at which participants switch to a different grip. This hypothesis was confirmed as the location of the switch point was affected by the surrounding frame. These findings extend observations of Glover and Dixon (2001b; Glover et al. 2005; see also Van Doorn et al. 2007), in which an orientation illusion was shown to affect grip choice in a simple grasping task. Second, our findings are in line with the predictions stemming from the perception-action model (Milner and Goodale 1995). Goodale and Milner propose that the ventral stream is responsible for “the perceptual representation of the perceptual world that is used in the planning of actions” (Goodale and Milner 2004, p. 38), thus assuming action planning to be subject to a visual illusion. Support for the hypothesis that the ventral stream plays an important role in action planning has also been reported in a patient study by Dijkerman et al. (2003), in which two patients with ventral stream lesions did not show appropriate switching when grasping bars in different orientations. Finally, our results can also be accommodated by the common-representation model of Franz (2001), in which it is proposed that a visual illusion affects both perception and action. It is important to note here that our study was not aimed at providing a critical test for one of these models. Rather, we aimed to examine what components of planning are affected by visual context.
The “rod-and-frame” illusion has been investigated by Dyde and Milner (2002), who found that the illusion influenced perception but not action. At first glance, these results may appear contradictory to our findings, but we argue that the difference in task constraints may have contributed to the different findings. As Smeets et al. (2002) argued, different tasks necessitate different types of spatial information to be used for action. For example in the Dyde and Milner study (2002) participants grasped the ends of the rod between their thumb and forefinger and participants were therefore dependent on the visual information regarding the position of the ends of the rod. In contrast, the orientation of the rod constituted the relevant action-related information source for participants in our study. Smeets and Brenner (1995) and Smeets et al. (2002) have proposed that an illusion only effects on action when the critical spatial characteristics of the target in the relation to the to-be-performed action are affected by the illusion. In our study this critical spatial characteristic was the orientation of the rod, whereas in the Dyde and Milner study it was the position of the ends of the rod.
Finally, two issues should be mentioned here, namely, the comparison between the perception task and the action task, and second, the unexpected finding of two strategy groups. The first issue concerns the comparison of illusion effects on perception and action. As Franz (2001) pointed out, an inherent problem in visual illusion studies is the comparison between the perception and the action task, as these tasks are predominantly measured by different methods, as was also the case in the present experiment. However, although our study does not allow us to compare perception and action in a quantitative way, the perception task did provide information about how participants perceived the rod orientation when surrounded by a tilted frame. Specifically, participants’ perception of the rod orientation was affected by the surrounding frame.
The second issue concerns the finding of two strategies. Our results showed that the means (i.e., grip type) by which the end goal was reached was affected by the visual illusion, however, participants reached the end goal differently, viz used different strategies. Most participants switched between pronated and supinated start postures, such that they ended the movement with a comfortable end posture. Still, three participants in our study did not obey this “end-posture comfort” rule. They used a pronated start posture that resulted in both uncomfortable and comfortable end postures. Importantly, however, irrespective of the strategy used, the effect of the visual context on grip planning was consistent. That is, tilting the frame affected the rod orientation where participants switched their grip, but depending on the strategy most participants switched between grip type 1 and 2 and some participants switched between grip type 1 and 3. This unexpected finding begs the question as to why some participants used a strategy that did not enable them to end the task in a comfortable end posture? The “posture based motion planning”-model of Rosenbaum et al. (2001) assumes that prior to movement execution an end posture is chosen from the stored posture base. The model further assumes a time constraint for this search process. If enough time is allowed, the search will most likely result in a posture that satisfies the end comfort criterium. If, however, insufficient time is allowed for the search, end postures may be selected that are not optimal. More specifically, these postures may be uncomfortable or even unfit for the task (see Meulenbroek et al. 2001 for model simulation and validation). In our study we instructed participants to perform the task “as fast as possible”. Therefore, it may be speculated that the “pronation starters” have put more emphasis on the speed of responding, thereby not completely searching their stored posture base. As the group of “pronation-starters” was small (n = 3), we cannot draw any definite conclusions on this matter but further examination is warranted.

visual illusion
motor planning
perceptual judgment
grip selection


Impact of headache in Europe: a review for the Eurolight project

A recent health economic survey in Europe has suggested that migraine is the costliest among the neurological disorders. According to many studies, migraine and other disorders lead to widespread suffering, reduction of quality of life, and marked impairment of participation, both in work and social activities. The present literature survey was made in order to summarize what is known on the subject, as a preparation for a EU-supported study to assess the impact in several EU countries with similar methodology and the same research instrument. Previous studies have yielded relatively reliable data only for migraine, whereas the impact of tension-type headache is virtually unknown or only very incompletely known for most dimensions of headache impact. Some data do suggest, however, that this headache may be as important from a health economic and a public health perspective as migraine. In future studies it is important to get population-based data from various countries relevant for estimation of indirect (mostly absenteeism from work and reduced working efficiency when having headache) and direct costs (related to medication, consultations, investigations and hospitalisations). Also, the impact on ability to get education and participate in the workforce is very relevant, as is the impact on love life and family planning. The quality of life of headache patients should be measured by validated instruments. To get a complete picture, one should also ask about the effect on the life of partners and children, and on the possible impact even when headache-free (e.g. fear of the next attack).
Introduction
For a just and rational distribution of means to health-care services and health-related research, reliable data on the individual and societal impact of different disorders are crucial. In recent years several initiatives have been launched to raise the awareness that headache is not only a nuisance for some individuals, but also that it entails widespread suffering and loss of opportunities for patients and their families, and large cost for the society. The recently published report on the prevalence and burden of headache [1] is a premise for the campaign “Lifting the burden: The Global Campaign to reduce the burden of headache”[2]. In Europe, much data on both the economic costs of migraine have been collected and presented in connection with the “Cost of Brain Disorders in Europe” project [3], in which migraine is treated along with many of the other (neurological and psychiatric) “brain disorders”. The Eurolight project (http://www.eurolight-online.eu) is an initiative supported by the EC Public Health Excecutive Agency launched in May 2007. Its objectives are to bring together the relevant medical, scientific and lay organizations, and to gather updated reliable comparable information regarding migraine, tension-type and chronic headache. It will be the first data collection on headaches at EU level focusing on a holistic, patient-driven and scientifically validated approach, aiming to fill in the main holes in our knowledge by performing comparable studies on headache prevalence and its impact in selected European countries (Austria, France, Germany, Italy, Lithuania, the Netherlands, Spain, UK, Ireland, Luxembourg). A pilot study has already been performed in Luxembourg. The present review of the existing literature on both economic and non-economic impact was performed as a part of the Eurolight project, to assess the current state of knowledge, and to build up a questionnaire to measure all the most relevant aspects of headache impact. This study presents the results of this review and a suggestion of the main dimensions that ought to be covered in the headache impact tool to be used in the Eurolight study.
Economic impact of headache
Headache may have considerable economic consequences, both for the patient and for the society as a whole. There are more studies about the societal costs than about the individual economic losses of the patients.
Relation to socioeconomic status, education and employment
In a large Norwegian study (the HUNT study), both migraine and headache in general was associated with low socioeconomic status [4], which has also been found in North America [5, 6] but not in some smaller European studies [7–11]. The question whether this is a consequence or a cause of headache is not satisfactorily answered, but in one Swedish study, half of the patients reported a negative influence of migraine on their ability to pursue studies and one third a negative influence on their finances [9]. In a US study it was found that headache patients have somewhat reduced labour force participation [12], but employment status has not been found to be related to headache in some European studies [7, 13].
Absenteeism from work
In two relatively old studies, one from Finland in 1979 [14] and one from San Marino in 1986 [15], 7% of working individuals had been absent from work in the previous year due headache. In a Danish study of 1992 [16], it was found that 43% of migraineurs (5% of the population) and 12% of TTH patients (9% of the population) had been absent from work during the previous year due to headache, i.e. a total of 14% of the population. In a Swedish study of 2004 [9], it was found that 65% of migraineurs reported some degree of absence from either school or work during the previous year. These data are, however, of relatively limited interest from an economical viewpoint as they do not indicate the number of days that the headache sufferers are away from work.
The number of days with work absence due to headache is relatively consistent across studies from different countries. In some previous studies it has varied between 2 and 6 days per year among headache patients in general [17], and between 1.5 and 4.2 days per year in migraineurs [9]. A study among migraineurs in Sweden revealed that 35% were never absent from work due to migraine, and 54% were absent 1–2 days per year [9]. Compared to headache-free individuals, migraine patients in the HUNT study from Norway lost on average 4.4 workdays per year, and persons with non-migrainous headache lost 2.5 workdays per year [18]. In the Danish study from Copenhagen [16], the TTH patients who had been absent seem to have been as much or more absent from work than the migraine patients, and the number of workdays lost due to migraine was 270 and to TTH 820 per 1,000 persons per year, i.e. a total of 1,090 days. In a study from England in 2003 [13], 15% had been absent from work or had reduced ability to work due to headaches in the previous 3 months. Per year, headache accounted for 1,327 missed and 5,213 reduced ability days per 1,000 workers per year, representing 0.5 and 2.0% of all working days in the adult population, irrespective of headache status. This study did not relate absenteeism to different headache diagnoses. In an English study of 2003, an estimated 5.7 workdays per year was missed by migraineurs working or attending school [8]. This seems to be higher than in France where a diary-based registration of absenteeism published in 1999 showed that migraineurs were away from work 2.18 days per year due to headache [19].
Effectiveness when working with headache
Working with migraine results in a 35% productivity loss on average according to some European studies [20]. This figure is, however, largely based on migraineurs’ self-report, which may give a too high estimate according to a recent US study from a workplace[21]. In this study, it was found that the working ability assessed by self-report was much lower than the objectively measured working efficiency (20 vs. 8%). The relatively small decline in working ability led the authors to conclude that workers with even relatively severe headache find creative ways to cope with the pain and maintain standards.
Health economic studies
For the headache part of the “Cost of Brain Disorders in Europe” project, a literature search for studies containing cost data for migraine and other headaches identified eight European studies evaluating the direct or indirect costs of migraine from a societal perspective [20], from France [22, 23], Germany [24], The Netherlands [25], Spain [26], Sweden [27] and the UK [28, 29]. No studies analysing the cost of TTH or other non-migraineous headaches were found. There were large variations in costs across the six European countries where data were available, ranging from around €100 per patient per year in Sweden to nearly €900 in Germany. These variations are probably mostly due to different methodologies and differences in the year when the studies were conducted. An important finding was that the vast majority of total costs, between 72 and 98%, was indirect costs, due to lost productivity, either in the form of work absence or reduced efficiency levels when working with migraine. Women tended to lose more workdays than men, but indirect costs were similar due to lower salaries and labour force participation amongst women. The direct costs, related to consultation, diagnostic investigations, treatments, and hospital admissions accounted for less than 30% of total costs in most studies.
The cost estimate for migraine in the European report was based on an average of the most representative cost estimates, from the UK, Germany and France. An average annual cost of €585 per migraine patient was estimated for these Western European countries. The 1-year prevalence of migraine was 14% among adults in Europe according to the review of epidemiological studies [1], i.e. 41 million adult Europeans with active migraine. Per patient migraine was the least costly disorder among the brain disorders. However, due to the high prevalence, the total cost of migraine was estimated to be €27 billion for whole Europe in 2004, which was the highest cost among the purely neurological disorders. Many of the psychiatric afflictions were even more costly according to this review. It is, however, likely that the available cost data in Europe would tend to underestimate the actual costs of headache, mainly because no cost data existed on the most common headache type (TTH), but also because children and adolescents were not considered, and because cost connected with more expensive medication (triptans) were not included, since most cost studies were performed before this class of drugs was introduced.
In a separate paper summarizing the prevalence and cost data for headache in Europe [30] a more speculative estimate for the cost of headache, rather than migraine alone, was derived by using the results of the Danish [31] and British [32] population-based studies which demonstrated that around 1,100–1,300 days per 1,000 workers were missed due to headache each year. The British study also suggested that the number of days with reduced efficacy was around four times higher than the number of days missed. Assuming a reduced efficiency of 35% when working with headache, and that the direct costs of headache constitute the same proportion of the total costs as for migraine, the average total cost per headache patient was estimated to be roughly €420 per year (of which €390 would be due to indirect costs and €30 due to direct medical costs). Since headache in general was found to affect nearly 50 % of Europeans, this estimate, if true, would make headache a much more costly disorder than migraine alone.
It is of interest to compare the European cost study [3] with more recent cost studies in some individual European countries. In one study from Spain [33] the annual costs of migraine was only about 50% of the sum given in the European Cost study for the same country. The difference may partly be explained by somewhat lower prevalence figures (12 vs. 14%) for migraine used in the Spanish study, but the main difference may be that this study did not employ a bottom-up design, but used published statistics and data to estimate resource use and productivity losses, which may have led to an underestimation of some costs. A recent study from France [34], restricted to the direct costs in 1999, found that these costs were at least twice as high (€128) as in the European migraine cost study (<€60). This study included both “strict” migraine (IHS 1.1 and 1.2) and “migraineous disorder” (IHS 1.7, corresponding to 1.6 in ICHD-2), which together affected 17% of the population. For the whole country the direct costs amounted to more than 1 billion €, which was 0.068% of the gross national product. Non-migraineous episodic headache, affecting 9.2% of the population, entailed a considerably lower cost of €28.
It may also be of interest to compare the European studies with one US study using a quite different methodology to assess direct costs. In this study, all types of medical care costs (not only those related to headache) were derived from the claims records of a large health plan, whereas diagnostic status (migraine or not) and comorbid and demographic status was ascertained using a telephone interview among members of the health plan [35]. Migraineurs incurred on average $700 more per year in total medical care costs than the controls. Interestingly, this statistically significant difference disappeared when psychiatric comorbidity variables (anxiety and depression) were entered into the model. The much higher costs per patient reflected in this study than in the French study [34] and Spanish study [33] may therefore at least partly be due to the differences in cost assessment methodology, indicating that the direct costs specifically related to migraine and not to comorbid disorders are most reliably assessed by a direct method, questioning patients about use of health-care resources.
Medication for headache constitutes an important part of the direct costs. In France in year 2000, the most frequently used acute medications for migraine were paracetamol, salicylates and NSAIDs. Triptans were used by 8% of migraineurs, and prophylactic treatment was used by 6% [36]. In Denmark 26% of migraineurs had used triptans in 2001, but less than 5% of those with pure migraine had used prophylactic medication [37].
Non-economic impact
From a purely humanitarian perspective, but also from a public health perspective, the pain, suffering and disability caused by headaches are as important as the economic consequences. In a study performed in young women in nine Western European countries, 86% of migraineurs stated that their life would have been better if they did not suffer form migraine [38]. A German study showed that, on average, patients with migraine or TTH had around 1 month every year affected by headaches [10]. The main burden of headache is carried by a minority of sufferers, and a Swedish study has shown that 27% of migraine patients had 68% of all attacks [9]. Three to 4% of the European population have headache half of the days or more per month [1].
Disability
It has been calculated that in the US, 300,000 persons stay in bed each day (24 h) due to headaches [39]. A Swedish study has shown that the disability is not only related to the attacks since many migraine patients report an impairment also between attacks [40]. Nine % of patients report that they have some residual disability since they do not recover completely between attacks, and in addition, many patients live in a constant worry about the next attack [9, 38].
In some studies, the level of disability due to migraine has been evaluated with the Migraine Disability Assessment Scale (MIDAS). With this instrument, days with work absence (job or household chores), days with ≥50% reduction in productivity, and days with inability to participate in social activities are counted during a 3-month period. In France, among those with active migraine, 22% (1.5% of the whole population) had grades III or IV disability (moderate or severe disability, indicating 11 days or more during the last 3 months’ period when headache affected work/household chores 50% or more, or leisure activities) [36]. MIDAS III or IV were about twice as common among migraineurs in one US study (54%) [41], as it was in the multinational Latin American study (50%) [42]. Among patients with headache in general (both migraine and non-migraineous headache, comprising 70% of the study population), 10.3% (7.2% of the population) had MIDAS grade III or IV disability [13]. Comparing the percentage of the general population in France with MIDAS disability grade III–IV due to migraine (1.5%) [36], with the percentage of population in England with same disability due to headache in general (7.2%) [13], it seems that non-migraineous headache causes more disability on a population than does migraine. The headache-attributed lost time (HALT) index is a close derivative of MIDAS (http://www.liftingtheburden.org/ → Resources → Burden measure) to be used for headache burden studies, which will be conducted by the Lifting The Burden Campaign [43].
According to the World Health Organisation (WHO), the preferred measure of disease burden is “Disability Adjusted Life Years” (DALYs), which is a sum of the years of life lost (YLL) and the years lived with disability (YLDs). The YLDs are determined by the incidence and duration of the disorder, and by a disability weight ranging between 0 and 1 [44]. Although migraine entails no increased mortality (i.e. YLL = 0), it ranked the 19th among the leading causes of DALYs among women aged between 15–44 years, and with regard to YLDs, it was 19th for both sexes, and the 12th for women, irrespective of age. Using the WHO data for a calculation of the burden of “brain disorders” (i.e. the psychiatric and neurological disorders) in Europe, the weight of migraine was lower than that of the major psychiatric disorders, dementias, stroke and injuries, but higher than that of epilepsy, multiple sclerosis and Parkinson’s disease[45].
In a recent report on the global prevalence and burden of headache disorders, the burden of migraine and TTH were measured in a similar way as the DALYs by combining data on prevalence, mean number and duration of headache attacks, and headache intensity, from studies containing such information. For the world as a whole, it was demonstrated that TTH resulted in a higher population burden (approximately 55% of total burden) than migraine (45%). The data for Europe indicated an even higher burden due to TTH compared to migraine [1]. If one uses the European data from this study it can be calculated that the hours with migraine headache would add up to between 34 and 100 h per year, if distributed on each adult individual in the population. The data on TTH are too scarce to use for similar calculations.
Studies using validated QoL-instruments
The SF-36 is a validated instrument to measure quality of life (QoL), containing eight dimensions. One US study [46], recruiting migraine patients from a medication trial, demonstrated that migraineurs had lower QoL than the general US population, most marked for bodily pain, physical role limitations and social functioning.
A Dutch population-based study found that migraine had a negative influence on all a dimensions compared to controls. The negative influence on QoL was larger than that of e.g. asthma, and it increased with increasing headache frequency [47]. Two population-based studies from Spain among chronic daily headache sufferers showed a marked negative influence, most marked for those with medication overuse, but similar for those with a headache of a migraine or a tension type [48]. One of these showed that the headache frequency may have a greater impact than headache intensity on QoL [48], and the other that chronic headache with medication overuse was associated with a decrease in all QoL aspects studied with SF-36, most marked for physical role and bodily pain [49]. A study from UK showed that migraineurs with high or moderate disability had a marked reduction on all dimensions on the SF-36 [50]. One Swedish study compared SF-36 results in the two sexes and in participants with different pain conditions. There was a gender difference for headache, which in men influenced physical function, physical role and bodily pain most, and in women vitality, social functioning, emotional functioning and mental health [51]. In a French study, migraineurs had significantly lower scores than headache-free controls on all SF-36 dimensions, and lower scores on the pain dimension than those with other headaches or with TTH [52].
One study comparing migraineurs in the US and the UK used a shorter QoL instrument, the SF-12, which contains a physical and a mental component [53]. In both countries, migraineurs had lower scores than controls on both components also after adjusting for socioeconomic status and for depression. However, in those with both migraine and depression, the QoL was significantly reduced in comparison to those who were not depressed.
In another French study, using a disease-specific QoL instrument called QVM, the QoL was found to be lowest among those with chronic headache, intermediate among migraineurs and highest among subjects with other forms of episodic headache [54].
The total burden of headache patients may not only be related to the headache per se, but also to comorbid conditions. European population-based studies have demonstrated that depression and/or anxiety occur two to three times more often among migraineurs than in the general population [55, 56]. Depression adds to the reduction in QoL in migraine [53]. This comorbidity may be as important for non-migrainous headache [57], but it is not known how this comorbidity influences the QoL in other headaches. In addition, it has been found that headache is also comorbid with other bodily pain, both in Finnish children [58] and Norwegian adults [59].
Family impact of migraine
Migraine also affects the patients’ spouses and children. In a population-based Swedish study [9], the percentage of migraine sufferers who reported a negative impact of migraine was 76% for attendance to work, 67% for family situation, 59% for leisure time, 48% for pursuing studies, 46% for sexual life, 37 % for their social position, 31% for love, 30% for their financial situation, 27% for making a career, and 11% for making friends.
One study has measured the impact on the family in two population samples of similar size in US and UK [60]. The impact was very similar in both countries. More than 60% of patients reported a marked impact on the ability to do household chores because of their migraine during the past 3 months, and it was markedly reduced also in 20% of the patients’ partners. Almost 46% of patients, and 24% of partners had missed days of family or social activities due to the proband’s migraine, and 16% of patients and 12% of partners had avoided making plans for family or social activities due to the proband’s migraine. As to the impact on the children of patients, more than 60% stated that it had a moderate to marked influence on the relation with their children, 40% stated that they would have been a better guardian or parent without migraine, more than 10% stated that their children had missed school, and 10% that their children had been late to school because of their headache. Forty-six percent of patients stated that they would have been better partners without headaches, and 5% stated that they had had fewer children because of headache, 0.4% that they had avoided having children, and 15% that they had avoided oral contraception. Compared with a control group, the partners of migraine patients were significantly more dissatisfied with the demands, responsibility and duties placed upon them, and with their ability to perform.
Conclusions
Health economic studies have documented that the costs of headache disorders are huge; the costs only for migraine amounting to €27 billion in the EU countries, and the cost for other headaches are probably as large. However, better population-based cost studies are needed to assess the cost involved with TTH. Headache sufferers tend to have lower income and education, and more of them may be unemployed, but it is still uncertain whether this is true for most European countries, and also whether it may be a cause of or an effect of headache. It is also amply documented that migraine confers a high degree of disability with more forced absence from work and leisure activities, and migraineurs also have a measurably reduced quality of life. In addition, there is a marked impact on family life, and headaches also put considerable strains on partners and children. A minority of headache sufferers chose to have fewer children than they would have had if they had not had headaches.
Based on the present review we have identified some main domains, summarized in the Table 1, that should be covered in order to capture as much as possible of the headache burden. The investigations should be performed in population-based samples, and to assess the whole burden, it is particularly important that not only migraine is included but also TTH and the chronic headaches. Different studies show marked variations between countries, both with regard to prevalence and burden of headache. At present it is not possible to determine with certainty whether these differences are real or due to variations in methodology. This highlights the need to study several countries with the same methodology and instrument, which will be done in the ongoing studies of the Eurolight. If further research shows that there are real and important differences between various countries and regions, investigations to determine the causes of such differences may elucidate ways to lower the burden of headache in a population. In any case, we believe that the Eurolight studies will provide the evidence needed to let headache disorders get the resources for treatment and research that they deserve according to the burden they place on people in Europe.
Table 1The main domains that should be covered in headache burden studiesEconomic burden Direct costMedication, consultation, investigations, hospitalisations Indirect costsWorkdays lostDecreased effectiveness when working with headacheLost career and education opportunitiesNon-economic burden DisabilityMIDAS or HALTTime with disability: Headache frequency x duration x intensity/disability Impact outside attacksResidual disability and fear of next attack Quality of LifeSF 36, SF 12, WHOQual etc Family impactImpact on the life of partner and/or childrenImpact on marriage and love lifeImpact on family planning and/or contraception Psychiatric complaintsVarious scales to measure anxiety and/or depression as consequent or comorbid disorders (e.g. HADS)

impact headache eurolight migraine burden disability


Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications.
Introduction
Recent years have seen a significant increase of interest in hydrophilic interaction liquid chromatography (HILIC). Although HILIC was first introduced in the 1970s [1], it took until the start of this century for a considerable number of HILIC applications to appear, highlighting its versatile nature [2–7]. A major cause of this increase is the growing need for the analysis of polar compounds that do not bind to reversed-phase (RP) materials and the constantly increasing complexity of samples [8]. Additionally, the exceptional growth of liquid chromatography–electrospray ionisation–mass spectrometry (LC–ESI–MS) for analytical analyses has led to a widening search for acceptable chromatographic materials. In this search it has been noted that the buffer conditions that are used for HILIC are highly compatible with MS, and that the high organic contents of these buffers can potentially increase sensitivity in ESI–MS[9, 10]. Moreover, an HILIC separation is “orthogonal” to RP separation, which makes it viable for the multidimensional separation of complex samples [11–13]. Finally, HILIC has been found to allow enrichment and the targeted analysis of post-translational modifications (PTMs) such as glycosylation [14], N-acetylation [12] and phosphorylation [15] in proteomics applications.
Although HILIC applications cover a large range of bioactive compounds, in this review we will focus on the use of HILIC in proteomics. We will describe how HILIC can be used for the separation of peptides in multidimensional chromatography and provide insights into its sensitivity, selectivity, separation power and “orthogonality.” We will also discuss how HILIC has recently been used for the enrichment of post-translational modifications at both peptide and protein levels.
HILIC
HILIC is characterized by the use of a hydrophilic stationary phase and a hydrophobic organic mobile phase [16], which was originally described by Linden et al. in 1975 [1]. The order of elution is reversed relative to reversed-phase chromatography (RP), with hydrophilic compounds being retained longer than hydrophobic compounds. Therefore, HILIC can simply be seen as a form of normal-phase (NP) chromatography. However, the acronym HILIC was suggested to distinguish it from NP, as NP is typically performed with nonaqueous, non-water-miscible solvent buffers, while HILIC is performed with water-miscible solvents and elution is achieved by a water gradient [2, 9, 17, 18].
In recent years, several stationary phases have emerged that are specifically made for HILIC approaches. Popular phases include underivatized silica stationary phases that contain functional groups such as siloxanes, silanols with (or without) a small quantity of metals [2, 9, 19, 20], derivatized silica, such as the cation exchanger polysulfoethyl A [17, 18], the weak cation exchanger Polycat A [21], the weak anion exchanger PolyWAX [22, 23], TSKgel amide 80 [24, 25], zwitterionic (ZIC)-HILIC [14] and “click” saccharides [26]; see Fig. 1 for structures of typical HILIC phases. Each of these materials display different retention characteristics and separation selectivities and require distinct buffer constitutions for optimal results [10].
Fig. 1Chemical structures of the functional groups in common HILIC stationary phases
A HILIC buffer typically contains more than 70% acetonitrile [17]. Other eluents have been tested, for instance methanol or isopropanol, but they resulted in poor chromatography or no analyte retention [2]. It is believed that the hydrophilic stationary phase enriches water from the buffer and thus generates an aqueous layer [17]. This allows for hydrophilic analytes to partition between the hydrophilic layer and the hydrophobic elution buffer. Elution is obtained through increasing the hydrophilicity of the mobile phase by increasing the water content. The final separation mechanism of elution, however, is most probably a superpositioning of partitioning and electrostatic interactions or hydrogen bonding to the stationary phase [2]. The extent to which each mechanism dominates is dependent on the actual type of stationary phase used and the buffer conditions, including the level and type of organic content, the type and concentration of salts and the pH [10]. Charged stationary phases, such as the abovementioned anion or cation exchangers and deprotonated silanol groups, are most likely to display some degree of electrostatic interaction [2]. Buffering salts in the mobile phase can decrease these electrostatic interactions through disruption. The choice of salts is however limited, due to the highly organic conditions of HILIC buffers, which makes it difficult to dissolve some salts in them [10]. Typically, ammonium acetate and formate are chosen because of their compatibility with MS, but ammonium bicarbonate, triethylamine phosphate (TEAP), sodium perchlorate and sodium methylphosphonate (Na-MePO4) have also been found to be applicable [2, 23]. Additionally, the same salt-based disruption can decrease the retention of analytes and can be useful during elution [17]. However, in some instances an increase in retention was seen upon increasing the salt concentration [10]. This was rationalized by the suggestion of a mechanism in which salt is enriched in the aqueous layer, which in turn increases the hydrophilicity of this liquid layer around the stationary phase [10].
Unsurprisingly, the type of salt that is used can also influence the retention behavior. It was observed that basic peptides were retained longer with TEAP in the buffer, while with Na-MePO4 these were the first peptides to elute with the weak anion exchanger PolyWAX as stationary phase [23]. It was reasoned that TEAP, as a counterion, will generate a sublayer where one negative charge of the salt is attracted to the positively charged stationary phase, while the second negative charge is free to attract basic peptide residues. In the case of Na-MePO4, there is only one negative charge that interacts with either the stationary phase or basic residues, leaving enough stationary phase available for interaction with acidic peptides [23].
A special type of HILIC called electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) actually specifically utilizes the electrostatic interactions in HILIC [23]. In ERLIC, a stationary phase is chosen that has a similar charge to the analytes to be separated. Analytes are on the one hand repelled by the stationary phase but on the other they are retained in the aqueous layer around the stationary phase. These opposing interactions allow isocratic resolution [23]. ERLIC can also be exploited in the selective isolation of phosphopeptides from a tryptic digest [23, 27].
Another factor that influences the retention characteristics in HILIC is the pH of the buffer. Whether the buffer pH is above or below the pKa of the analyte determines its charge state, which in turn affects the hydrophilicity of the analyte and likewise the interaction with the stationary phase [10, 12]. For example, large differences were found in the retention profiles of a highly complex tryptic digest run over the same column at pH 3 and pH 8 [12]. At pH 3, acidic peptide residues are protonated and so the overall hydrophilicity of peptides containing these residues is decreased, leading to poorer retention of these peptides, with the effect being magnified with higher numbers of acidic residues [12].
For a more detailed overview of different HILIC stationary phases and their applications outside the field of proteomics, see Hëmstrom et al. [2].
HILIC in multidimensional peptide separation
Shotgun proteomics involves the analysis of entire proteomes in single experiments. Analysis of such samples raises a number of issues, including enormous complexity, large dynamic range and low levels of available analyzable material. Often, SDS PAGE-based [28] or LC separation techniques are used to reduce the sample complexity, followed by RP LC-MS as the final readout. Major advances have been made in the miniaturization of LC–MS, since reducing the flow rate improves mass spectrometric sensitivity [29], reduces nonspecific adsorption onto separation devices, and improves chromatographic resolving power [30], but improvements are still required. The complexity of a sample can be further reduced prior to mass spectrometric analysis by the addition of extra dimensions of separation. At present, the method of choice is the combination of strong cation exchange (SCX) with RP [31–34]. Over time, a number of alternative configurations have been developed, including size exclusion chromatography (SEC)-RP [35, 36] and RP at pH 10 followed by RP at pH 2.6 [37]. However, the low separation power of SEC [36] and the correlative separation of RP at different pH levels has not yielded the optimal two-dimensional (2D) LC system yet.
Recently, HILIC was introduced as a dimension for 2D-LC–MS and was shown to remove a few of the disadvantages of existing techniques [11, 12]. Evidently, RP and HILIC buffers are not directly compatible and so online hyphenation of these stationary phases will be difficult. Online hyphenation possesses certain advantages, such as minimal loss of sample, no vial contamination, and no sample dilution [38, 39]. However, performing an experiment in an offline fashion is not necessarily inferior since overloading of the second dimension is no longer an issue with offline fractionation as it is for online approaches. Moreover, in an offline mode, columns and/or buffers that are normally not compatible with the two consecutive separations can be used [38, 40]. Finally, a conventional gradient for peptide separation can be used in offline setups, and this has been shown to be generally superior to step elution [40].
Gilar et al. separated and analyzed a mixture of approximately 200 peptides using different stationary phases, including SEC, HILIC, RP at pH 2.6 and pH 10 and SCX. HILIC was shown to have a separation power superior to both SCX and SEC [11]. The orthogonality—i.e., how different the separation mechanisms are—of the stationary phases was determined by plotting the peptide retention times of two dimensions against each other (see Fig. 2). Although this study did not actually experimentally connect the different stationary phases, since all LC–MS runs were one dimensional, it revealed that HILIC is slightly more orthogonal to RP than SCX [11].
Fig. 2Two-dimensional plots of normalized peptide retention times in HILIC, SCX and RP separation. Both HILIC and SCX have separation mechanisms that are orthogonal to RP, but the clustering of similarly charged peptides in SCX makes this a less optimal first dimension. From the work of Gilar et al. [11] and reproduced with permission from the American Chemical Society
A genuine two-dimensional HILIC–RP system was presented recently [12]. A microliter flow-scale ZIC–HILIC system was connected offline with a nanoliter flow-scale RPLC–MS. For semi-automation, a microliter-scale fraction collector was connected to the HILIC output to mix the eluent with RP- and MS-compatible aqueous buffer and to allow direct consecutive analysis of the HILIC fractions by MS. In this study, a level of orthogonality to RP was found that matched the theoretical picture presented by Gilar et al. [11]. SCX separation can be limited by the fact that similarly charged peptides cluster in narrow elution windows (see also Fig. 2), and it was shown that HILIC did not exhibit this behavior [11, 12].
Analysis of data acquired using this two-dimensional system shed further light on how HILIC can be orthogonal to RP. Indeed, a separation mechanism was observed that is based on both retention by hydrophilicity due to partitioning with the aqueous sublayer and retention by charge due to electrostatic interaction with the stationary phase [11, 12]. These electrostatic interactions ensure that HILIC separation is more than merely the reverse of RP, while the presence of hydrophilic interactions made similarly charged peptides elute over a wider window [12].
It was further noted that the orthogonality of ZIC–HILIC with RP is dependent on the buffer pH. At pH 3, a higher orthogonality was seen than at pH 6.8 and pH 8 [12].
The studies discussed so far were all performed with HILIC columns packed with particles. A further improvement in separation could be obtained by using monolithic structures instead of porous particles. The higher porosity of monolithic materials enables the use of longer columns and/or higher flow rates, increasing the separation efficiency without increasing the backpressure [41]. Recently, attempts to develop HILIC columns that possess monolithic characteristics have been reported [41–45]. Horie et al. demonstrated the use of HILIC monoliths for peptide separations [42]. The authors applied increased flow rates and confirmed that short gradients of 3–10 min were possible with 20-cm-long 200 μm ID columns, while the peak capacities were comparable to a similar monolithic RP setup. The poor peptide sequencing speed of a mass spectrometer can be a potential drawback in the direct coupling of such columns to MS. Currently, mass spectrometers can sequence 2–3 peptides per second, which is significantly lower than the number of peptides that will be delivered by the monolith column. However, such columns are highly applicable as a first dimension, where time is not a parameter.
HILIC is proving to be an attractive choice among the range of separation methods available for the proteomics researcher. The combinations chosen so far for 2D-LC–MS, as reported in the literature, have been limited to HILIC–RP, but the versatility of the chromatographic material will also allow combinations such as SCX–HILIC and three-dimensional approaches can be envisaged. The high organic levels utilized in HILIC buffers compared to RP buffers have the additional benefit that they improve electrospray ionization, which increases the sensitivity of ESI–MS [9], thus suggesting that it may be an interesting final dimension [11].
HILIC for the analysis of PTMs
The analysis of protein PTMs is a very important albeit challenging task in proteomics. PTMs are generally present at much lower levels than their unmodified counterparts, which makes it very easy to “miss them” in untargeted analyses, such as those performed using shotgun proteomics techniques [46]. Protein modifications such as phosphorylation and glycosylation have proven to be difficult to investigate in a routine fashion. This is mainly because these moieties are prone to elimination and adsorption if care is not taken with sample preparation and MS analysis protocols [14, 47]. Recently, some of these MS problems have been overcome by using alternative MS peptide fragmentation techniques, such as electron capture dissociation (ECD) [48] and electron transfer dissociation (ETD) [49]. Several approaches have been developed to counter some of the problems presented by the low abundance and highly labile nature of PTM peptides. The use of chelation [50–52] or specific chromatographic materials [47] have proven to be successful strategies, and in the latter category HILIC has proven to be a worthy addition.
HILIC in the targeted analysis of phosphorylated peptides
Phosphorylation is probably the most studied protein post-translational modification in proteomics; it provides an important function in signal transduction, metabolic maintenance and cell division [53]. The development of techniques to enrich for phosphopeptides has involved major efforts in phosphoproteomics. One of the more successful approaches targets the enrichment of phosphopeptides by SCX at low pH [47]. At pH 3, acidic residues such as glutamic and aspartic acid are neutral while phospho-serine/threonine/tyrosine will still be negative. Such a distinction allows proteolytic tryptic phosphopeptides to be separated from “normal” tryptic peptides due to an earlier elution. However, other peptide subgroups with reduced net charge, such as N-acetylated tryptic peptides, will also exhibit a shorter retention time, and thus a second level of enrichment is required to separate coeluting nonphosphorylated peptides from phosphorylated peptides [54]. Furthermore, the trypsinization of proteins is less efficient for regions where phosphomoieties are present [55]. Thus a large fraction of proteolytic phosphopeptides will contain miscleavages (i.e., additional basic residues), and will not be enriched. Therefore, other techniques for the targeted enrichment of phosphopeptides are required and are generally performed after SCX, such as those based on immobilized metal affinity chromatography (IMAC) [50, 54] or the metal oxide TiO2 [51, 52, 56]. One other limitation of SCX for phosphopeptide enrichment is that it is based on poor retention. This means that multiply phosphorylated peptides, another large subgroup, are difficult to retain or are even lost. The use of strong anion exchange (SAX) stationary phases immediately following SCX retention can circumvent some of these problems [57]. The generally lower pI values of phosphopeptides will allow stronger retentions on SAX than “normal” peptides. However, the extra LC separation increases the analysis time and the likelihood of sample loss.
Finally, a mixed-bed ion exchange method with both cation and anion exchange was presented recently and was shown to recover more normal tryptic peptides than SCX and to exhibit orthogonal separation towards RP [58]. Phosphopeptides could be eluted by applying a single final salt step. However, in this step only a subset of phosphopeptides—the acidic ones—could be resolved.
HILIC is presented as an alternative enrichment technique, as it exploits the increase in hydrophilicity of peptides through the attachment of a phosphogroup [15, 22, 23, 27]. McNulty et al. applied a TSKgel Amide-80 based HILIC system to the phosphopeptide analysis of a Calyculin A-treated HeLa cell lysate digest. The authors demonstrated that the system, under optimal conditions, allowed phosphorylated peptides to be separated from nonphosphorylated peptides and acidic peptides that might interfere with subsequent IMAC enrichment. Interestingly, the phosphopeptides eluted in the middle of the HILIC separation and would also allow a considerable level of fractionation. This rudimentary analysis permitted more than 1000 unique phosphopeptides to be identified after further IMAC enrichment and LC–MS analysis [15].
Ytterberg et al. evaluated the use of HILIC in a preparative separation set-up using HILIC material packed in micro tips [22]. Different combinations of SCX, RP and two types of HILIC [polyhydroxyethyl aspartamide (PHEA) and PolyWAX] were tested for the enrichment of phosphopeptides from a saliva digest. As expected, most of the phosphorylated peptides were retained strongly under typical HILIC analysis conditions. Furthermore, the separation power of the stepwise elution was sufficient to separate peptides with differing numbers of phosphorylated residues. The conditions used with PolyWAX are ERLIC conditions and seem to be very useful for phosphopeptide enrichment [23]. Normal tryptic peptides are simultaneously repelled by the weak anion exchange material and attracted by hydrophilic interactions, while phosphopeptides are attracted by both. This mode of separation is very responsive to salt contents and the pH of the buffer and can thus be tweaked to enrich for highly phosphorylated peptides containing up to six phosphorylated residues [22].
In contrast to these results obtained with TSKgel Amide-80, PHEA and PolyWAX, the enrichment of phosphopeptides was not observed with ZIC–HILIC [12]. This might be explained by the zwitterionic nature of this chromatographic material: the negative charge could potentially repel the phosphate group, resulting in phosphopeptides eluting with nonphosphorylated peptides.
The microcolumn approach is a simple and undemanding preparative method. However, HPLC-mode HILIC has the potential for automation and fractionation, as the HILIC buffers are directly compatible with IMAC and TiO2. It might be a valuable alternative to SCX, with improved fractionation and better retention of multiply phosphorylated peptides, and so it potentially could allow a more comprehensive analysis of the phosphoproteome. While it should be noted that the application of HILIC to phosphoproteomics is still in its infancy, it is likely that the actual power of HILIC in this field will become clear in the next few years.
HILIC in the targeted analysis of N-terminally acetylated peptides
The positive N-terminal charge of a protein can be neutralized by different post-translational modifications, each potentially influencing protein function, stability and interaction with other biomolecules [59]. Amongst the biologically relevant N-terminal modifications are acetylation, methylation and myristoylation [60]. As mentioned above, tryptic N-acetylated peptides can be enriched by SCX [33, 54, 61]. The acetylation neutralizes the N-terminal charge, lowering the net charge compared to the unmodified counterpart. Therefore, N-acetylated tryptic peptides will, like phosphopeptides, elute in the first few SCX fractions.
ZIC–HILIC has been proposed as another means of enriching N-acetylated tryptic peptides [12]. When it contains a neutralized N-terminus, the hydrophilicity of an N-acetylated peptide is decreased. This reduction in polarity is even more pronounced at pH 3, where only basic peptide residues are charged and acidic residues are protonated. Through the use of a ZIC–HILIC separation and fractionation, Boersema et al. showed that this subgroup of trypsinized peptides that are N-terminally blocked could be enriched due to their shorter retentions than “normal” trypsinized peptides (see Fig. 3) [12]. Similarly, other subgroups of N-terminally modified peptides, such as those that contain an N-terminal pyroglutamic acid, were also found primarily in the first ZIC–HILIC fractions [12].
Fig. 3Distribution of phosphorylated and N-acetylated peptides over ZIC–HILIC fractions. First dimension: ZIC-HILIC, 200 μm × 160 mm, 3.5 μm, 200 Å. Flow rate 1.5 μL/min, 1 min.fractions. Number of peptides: bare line, total; squares, N-acetylated; triangles, phosphorylated. ZIC–HILIC provides clear enrichment of N-acetylated peptides in the initial fractions. From the work of Boersema et al. [12], reproduced with permission from the American Chemical Society
Unfortunately, as trypsin cannot cleave acetylated lysine residues, peptides containing this modification could not be enriched. The miscleavage-containing K-acetylated tryptic peptide will possess a net charge that is the same as “normal” tryptic peptides [12].
Interestingly, and in contrast with SCX [33], no significant enrichment of C-terminal peptides was observed in this ZIC–HILIC set-up, although the hydrophilicity of these peptides is also different from “normal” tryptic peptides, due to the absence of a terminal arginine or lysine residue. It was hypothesized that this might be explained by the difference in charge distribution after losing an N-terminal positive charge compared to lacking a C-terminal positive charge. A C-terminal peptide would as a result be more polar—and thus more hydrophilic—compared to an N-terminally blocked peptide [12]. As suggested above, the ZIC–HILIC method will enrich for any peptide containing a neutralized N-terminus and thus would be applicable as a first step in the enrichment of formylated, carboxylated, hydroxylated, palmitoylated and myrisotylated peptides.
HILIC in the targeted analysis of glycosylated peptides
Glycosylation, the attachment of a carbohydrate moiety to a protein, is a frequent but very heterogeneous PTM. It modulates the physicochemical and biological properties of proteins and serves as a recognition determinant between molecules, molecules and cells, or between cells [62]. Glycosylation of outer cell wall proteins are, for instance, involved in host–pathogen interactions. It has been estimated that more than half of all proteins in a biological system are glycoproteins [63]. The heterogeneity of glycan structures makes a global proteomics characterization of glycosylation extremely difficult, as the signal intensity of glycosylated peptides with the same amino acid backbone is dispersed over peptides with different oligosaccharide chains [64]. The proteomics analysis of glycopeptides is made even more tedious by the fact that MS/MS data rarely allow the confident identification of peptide sequences due to the relatively large molecular weights of glycosylated peptides [65] and the complex fragmentation that originates from both peptide and glycan cleavages [14]. To overcome some of these issues in proteomics analysis, the labile sugar groups are often removed by deglycosylation prior to MS analysis [66]. The problem of low glycopeptide abundance can be tackled by enrichment techniques, mostly those based on lectin-mediated affinity capture [66]. Recently, HILIC was introduced as a promising additional enrichment step for glycopeptides [14].
The hydrophilicity of glycopeptides makes them ideal candidates for separation by HILIC. A first glycopeptide application for HILIC was found in the analysis of differentially sialylated glycopeptides from interferon-γ [67]. Fractions of glycopeptides separated by RP were further separated by HILIC. Their orthogonal retention on the HILIC column was shown to correlate well with the number of sialyl groups. Further studies by ESI–MS revealed that the interaction of N-glycans with ZIC–HILIC is based on a partitioning mechanism, while the separation of differently sialylated N-glycans could be explained by an electrostatic repulsion interaction mechanism [68]. Recently, more global proteomics approaches were reported that targeted protein glycosites using HILIC to enrich glycopeptides [14, 66, 69–72]. Generally, in these approaches glycoproteins were first selectively captured by lectin-mediated affinity chromatography, followed by SDS-PAGE and in-gel digestion. The peptides obtained were then further enriched for glycosylation using ZIC–HILIC microcolumns. The glycopeptides that were primarily found in the later fractions were then enzymatically degycosylated prior to MS analysis. Figure 4 shows the relative intensities after solid-phase extraction (SPE) of a tryptic digest of TIMP-1 with RP (R2) and HILIC microcolumns. The relative abundance of glycopeptides was significantly increased following enrichment by HILIC. SPE by RP caused the matrix-assisted laser desorption/ionization (MALDI) spectrum to be dominated by nonglycosylated peptides, while, in comparison, SPE with HILIC resulted in a spectrum crowded with glycosylated peptides [70].
Fig. 4MALDI-TOF spectra after SPE of a tryptic digest of TIMP-1 by RP (R2, upper spectrum) and HILIC (lower spectrum) microcolumns. HILIC clearly enriches for glycopeptides. This figure was kindly provided by Dr. P. Højrup (similar to [70])
Small diversions from this general protocol have been reported and include lectin-mediated affinity capture at the peptide level [69] and the further separation of glycopeptides by SCX [66]. It was shown that the unambiguous localization of glycosylation sites is facilitated by leaving a single GlcNac residue on the site [14] or by tagging the site using 18O isotope labeling [66, 69].
Using the HILIC method, 62 glycosylation sites could be identified for 37 glycoproteins in human plasma [14], while a further study of the Cohn IV fraction of human plasma revealed 103 N-glycosylation sites and 23 fucosylated N-glycosylation sites [66]. 1465 N-glycosylated sites were found on 829 proteins in C. elegans [72]. However, this study was performed using three 50–200 mg samples for affinity chromatography on three different lectin columns, and each LC–MS analysis was performed in triplicate [72], unlike the Cohn IV work, where a 1 mg fraction was used [66].
The glycosylation sites were determined in these studies, but not the actual glycan structure. A few studies have used HILIC for the desalting and enrichment [65, 70] or the separation [73] of intact glycopeptides. However, these studies all used relatively simple protein mixtures or even single proteins, indicating that the unambiguous characterization of peptide glycan structures at the proteome level is still rather challenging.
These early experiments show that HILIC can be used as an effective and relatively simple tool for the targeted analysis of protein glycosylation. Future studies are likely to follow, wherein HILIC might also have an important role to play in not only glycosite elucidation but also in the compositional analysis of glycan structures, as HILIC was already found to be convenient for the desalting and enrichment [64] or even the separation [74] of glycans themselves.
HILIC in the targeted analysis of histone modifications
The regulation of the chromatin structure, and therefore DNA transcription and replication, is driven by post-translational modifications of the core histones. These modifications, including methylation, acetylation, phosphorylation, sumoylation and ubiquitination, are primarily found at the histone N-terminal tails [75]. Different modifications can occur at the same histone, and it is emerging that not just single site modification but rather the interdependence of these modifications is very important to regulation [76]. The analysis of histone modifications on intact histone proteins by MS is very demanding, due to the enormous heterogeneity of the modifications [77]. The separation of differentially modified proteins prior to MS is therefore essential.
HILIC has been used in the last decade for the analysis of acetylated and methylated histones [5, 7, 21, 78–80]. In these studies, RP and HILIC phases are generally combined to separate different histones. In analyses where LC–MS analysis is used for the characterization of histone modifications, this is performed on the peptide level. However, information on the interdependence of modifications is lost due to the hydrolysis step, because different modifications will end up at different peptides [5, 6, 79–81]. To preserve this information, a HILIC separation of intact histones was recently performed using a top-down MS approach, i.e., at the level of intact proteins [77, 82]. Histone H4 was purified by RP while HILIC was used for the further separation of differentially modified forms. Subsequently, these H4 HILIC fractions were analyzed by ESI–Q–FTMS/MS with ECD fragmentation [82]. A similar but semi-top-down approach was performed for the analysis of H3.2. As H3.2 is not very soluble in the highly organic HILIC buffers, GluC digestion was employed to generate a 50 amino acid-long N-terminal peptide that contains most of the modification sites [77]. Figure 5 clearly shows the separation power of HILIC in the analysis of histones. It simultaneously separates acetylated and methylated histones, greatly reducing the complexity of the sample. Acetylation is the major determinant of separation. The separation of proteins for which acetylation was induced by butyrate resulted in more peaks at lower retention times. The smaller peaks within these larger peaks represent proteins with different levels of methylation.
Fig. 5HILIC separation of GluC-generated long N-terminal peptides from histone H3.2. HILIC primary separation is controlled by the number of acetylations (the three bigger peaks for the +butyrate sample), and within these peaks a secondary separation is observed, relating to the number of methylations (the narrower peaks). Adapted by permission from Macmillan Publishers Ltd: Nature Methods [77]
In these histone analyses, HILIC delivered the extra separation step that was needed to sufficiently reduce the complexity of the samples and to separate the most important histone modifications.
Concluding remarks
Recent years have witnessed an increased interest in HILIC. More versatile and diverse stationary phases have become available, leading to reports of an exciting and broad range of applications. The unique separation and orthogonality of HILIC towards RP (the most commonly used peptide separation method) make it an ideal method for multidimensional chromatography that can extend separation power. As far as selectivity is concerned, HILIC can compete well with RP and SCX, which are the two main chromatographic techniques applied today. Focusing solely on proteomics applications, HILIC has been shown to be very versatile in analyses of protein modifications. The retention of hydrophilic compounds can be effectively exploited in the enrichment of phosphorylated, N-terminally blocked and glycosylated peptides. Moreover, in combination with RP it allows the separation of complex differentially modified histones, an incredibly challenging area. It is therefore expected that the number of applications of HILIC in the proteomics field will increase significantly in the years to come, and that the development of novel HILIC stationary phases and/or monolithic columns will continue. Much progress is expected.

hilic
proteomics
post-translational modification
two-dimensional liquid chromatography