Hydrophilic interaction liquid chromatography -LRB- HILIC -RRB- 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 -LRB- SCX -RRB- and reversed-phase -LRB- RP -RRB- chromatography is the most widely used combination for the separation of peptides . Here , we review the potential of hydrophilic interaction liquid chromatography -LRB- HILIC -RRB- 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 -LRB- HILIC -RRB- . Although HILIC was first introduced in the 1970s -LSB- 1 -RSB- , it took until the start of this century for a considerable number of HILIC applications to appear , highlighting its versatile nature -LSB- 2 -- 7 -RSB- . A major cause of this increase is the growing need for the analysis of polar compounds that do not bind to reversed-phase -LRB- RP -RRB- materials and the constantly increasing complexity of samples -LSB- 8 -RSB- . Additionally , the exceptional growth of liquid chromatography -- electrospray ionisation -- mass spectrometry -LRB- LC -- ESI -- MS -RRB- 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 -LSB- 9 , 10 -RSB- . Moreover , an HILIC separation is `` orthogonal '' to RP separation , which makes it viable for the multidimensional separation of complex samples -LSB- 11 -- 13 -RSB- . Finally , HILIC has been found to allow enrichment and the targeted analysis of post-translational modifications -LRB- PTMs -RRB- such as glycosylation -LSB- 14 -RSB- , N-acetylation -LSB- 12 -RSB- and phosphorylation -LSB- 15 -RSB- 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 -LSB- 16 -RSB- , which was originally described by Linden et al. in 1975 -LSB- 1 -RSB- . The order of elution is reversed relative to reversed-phase chromatography -LRB- RP -RRB- , with hydrophilic compounds being retained longer than hydrophobic compounds . Therefore , HILIC can simply be seen as a form of normal-phase -LRB- NP -RRB- 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 -LSB- 2 , 9 , 17 , 18 -RSB- . 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 -LRB- or without -RRB- a small quantity of metals -LSB- 2 , 9 , 19 , 20 -RSB- , derivatized silica , such as the cation exchanger polysulfoethyl A -LSB- 17 , 18 -RSB- , the weak cation exchanger Polycat A -LSB- 21 -RSB- , the weak anion exchanger PolyWAX -LSB- 22 , 23 -RSB- , TSKgel amide 80 -LSB- 24 , 25 -RSB- , zwitterionic -LRB- ZIC -RRB- - HILIC -LSB- 14 -RSB- and `` click '' saccharides -LSB- 26 -RSB- ; 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 -LSB- 10 -RSB- . Fig. 1Chemical structures of the functional groups in common HILIC stationary phases A HILIC buffer typically contains more than 70 % acetonitrile -LSB- 17 -RSB- . Other eluents have been tested , for instance methanol or isopropanol , but they resulted in poor chromatography or no analyte retention -LSB- 2 -RSB- . It is believed that the hydrophilic stationary phase enriches water from the buffer and thus generates an aqueous layer -LSB- 17 -RSB- . 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 -LSB- 2 -RSB- . 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 -LSB- 10 -RSB- . 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 -LSB- 2 -RSB- . 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 -LSB- 10 -RSB- . Typically , ammonium acetate and formate are chosen because of their compatibility with MS , but ammonium bicarbonate , triethylamine phosphate -LRB- TEAP -RRB- , sodium perchlorate and sodium methylphosphonate -LRB- Na-MePO4 -RRB- have also been found to be applicable -LSB- 2 , 23 -RSB- . Additionally , the same salt-based disruption can decrease the retention of analytes and can be useful during elution -LSB- 17 -RSB- . However , in some instances an increase in retention was seen upon increasing the salt concentration -LSB- 10 -RSB- . 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 -LSB- 10 -RSB- . 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 -LSB- 23 -RSB- . 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 -LSB- 23 -RSB- . A special type of HILIC called electrostatic repulsion -- hydrophilic interaction chromatography -LRB- ERLIC -RRB- actually specifically utilizes the electrostatic interactions in HILIC -LSB- 23 -RSB- . 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 -LSB- 23 -RSB- . ERLIC can also be exploited in the selective isolation of phosphopeptides from a tryptic digest -LSB- 23 , 27 -RSB- . 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 -LSB- 10 , 12 -RSB- . 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 -LSB- 12 -RSB- . 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 -LSB- 12 -RSB- . For a more detailed overview of different HILIC stationary phases and their applications outside the field of proteomics , see Hëmstrom et al. -LSB- 2 -RSB- . 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 -LSB- 28 -RSB- 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 -LSB- 29 -RSB- , reduces nonspecific adsorption onto separation devices , and improves chromatographic resolving power -LSB- 30 -RSB- , 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 -LRB- SCX -RRB- with RP -LSB- 31 -- 34 -RSB- . Over time , a number of alternative configurations have been developed , including size exclusion chromatography -LRB- SEC -RRB- - RP -LSB- 35 , 36 -RSB- and RP at pH 10 followed by RP at pH 2.6 -LSB- 37 -RSB- . However , the low separation power of SEC -LSB- 36 -RSB- and the correlative separation of RP at different pH levels has not yielded the optimal two-dimensional -LRB- 2D -RRB- 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 -LSB- 11 , 12 -RSB- . 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 -LSB- 38 , 39 -RSB- . 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 -LSB- 38 , 40 -RSB- . 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 -LSB- 40 -RSB- . 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 -LSB- 11 -RSB- . 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 -LRB- see Fig. 2 -RRB- . 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 -LSB- 11 -RSB- . 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. -LSB- 11 -RSB- and reproduced with permission from the American Chemical Society A genuine two-dimensional HILIC -- RP system was presented recently -LSB- 12 -RSB- . 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. -LSB- 11 -RSB- . SCX separation can be limited by the fact that similarly charged peptides cluster in narrow elution windows -LRB- see also Fig. 2 -RRB- , and it was shown that HILIC did not exhibit this behavior -LSB- 11 , 12 -RSB- . 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 -LSB- 11 , 12 -RSB- . 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 -LSB- 12 -RSB- . 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 -LSB- 12 -RSB- . 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 -LSB- 41 -RSB- . Recently , attempts to develop HILIC columns that possess monolithic characteristics have been reported -LSB- 41 -- 45 -RSB- . Horie et al. demonstrated the use of HILIC monoliths for peptide separations -LSB- 42 -RSB- . 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 -LSB- 9 -RSB- , thus suggesting that it may be an interesting final dimension -LSB- 11 -RSB- . 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 -LSB- 46 -RSB- . 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 -LSB- 14 , 47 -RSB- . Recently , some of these MS problems have been overcome by using alternative MS peptide fragmentation techniques , such as electron capture dissociation -LRB- ECD -RRB- -LSB- 48 -RSB- and electron transfer dissociation -LRB- ETD -RRB- -LSB- 49 -RSB- . 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 -LSB- 50 -- 52 -RSB- or specific chromatographic materials -LSB- 47 -RSB- 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 -LSB- 53 -RSB- . 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 -LSB- 47 -RSB- . 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 -LSB- 54 -RSB- . Furthermore , the trypsinization of proteins is less efficient for regions where phosphomoieties are present -LSB- 55 -RSB- . Thus a large fraction of proteolytic phosphopeptides will contain miscleavages -LRB- i.e. , additional basic residues -RRB- , 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 -LRB- IMAC -RRB- -LSB- 50 , 54 -RSB- or the metal oxide TiO2 -LSB- 51 , 52 , 56 -RSB- . 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 -LRB- SAX -RRB- stationary phases immediately following SCX retention can circumvent some of these problems -LSB- 57 -RSB- . 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 -LSB- 58 -RSB- . 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 -LSB- 15 , 22 , 23 , 27 -RSB- . 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 -LSB- 15 -RSB- . Ytterberg et al. evaluated the use of HILIC in a preparative separation set-up using HILIC material packed in micro tips -LSB- 22 -RSB- . Different combinations of SCX , RP and two types of HILIC -LSB- polyhydroxyethyl aspartamide -LRB- PHEA -RRB- and PolyWAX -RSB- 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 -LSB- 23 -RSB- . 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 -LSB- 22 -RSB- . In contrast to these results obtained with TSKgel Amide-80 , PHEA and PolyWAX , the enrichment of phosphopeptides was not observed with ZIC -- HILIC -LSB- 12 -RSB- . 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 -LSB- 59 -RSB- . Amongst the biologically relevant N-terminal modifications are acetylation , methylation and myristoylation -LSB- 60 -RSB- . As mentioned above , tryptic N-acetylated peptides can be enriched by SCX -LSB- 33 , 54 , 61 -RSB- . 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 -LSB- 12 -RSB- . 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 -LRB- see Fig. 3 -RRB- -LSB- 12 -RSB- . 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 -LSB- 12 -RSB- . 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. -LSB- 12 -RSB- , reproduced with permission from the American Chemical Society Unfortunately , as trypsin can not 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 -LSB- 12 -RSB- . Interestingly , and in contrast with SCX -LSB- 33 -RSB- , 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 -LSB- 12 -RSB- . 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 -LSB- 62 -RSB- . 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 -LSB- 63 -RSB- . 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 -LSB- 64 -RSB- . 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 -LSB- 65 -RSB- and the complex fragmentation that originates from both peptide and glycan cleavages -LSB- 14 -RSB- . To overcome some of these issues in proteomics analysis , the labile sugar groups are often removed by deglycosylation prior to MS analysis -LSB- 66 -RSB- . The problem of low glycopeptide abundance can be tackled by enrichment techniques , mostly those based on lectin-mediated affinity capture -LSB- 66 -RSB- . Recently , HILIC was introduced as a promising additional enrichment step for glycopeptides -LSB- 14 -RSB- . 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 -LSB- 67 -RSB- . 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 -LSB- 68 -RSB- . Recently , more global proteomics approaches were reported that targeted protein glycosites using HILIC to enrich glycopeptides -LSB- 14 , 66 , 69 -- 72 -RSB- . 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 -LRB- SPE -RRB- of a tryptic digest of TIMP-1 with RP -LRB- R2 -RRB- 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 -LRB- MALDI -RRB- spectrum to be dominated by nonglycosylated peptides , while , in comparison , SPE with HILIC resulted in a spectrum crowded with glycosylated peptides -LSB- 70 -RSB- . Fig. 4MALDI-TOF spectra after SPE of a tryptic digest of TIMP-1 by RP -LRB- R2 , upper spectrum -RRB- and HILIC -LRB- lower spectrum -RRB- microcolumns . HILIC clearly enriches for glycopeptides . This figure was kindly provided by Dr. P. Højrup -LRB- similar to -LSB- 70 -RSB- -RRB- Small diversions from this general protocol have been reported and include lectin-mediated affinity capture at the peptide level -LSB- 69 -RSB- and the further separation of glycopeptides by SCX -LSB- 66 -RSB- . It was shown that the unambiguous localization of glycosylation sites is facilitated by leaving a single GlcNac residue on the site -LSB- 14 -RSB- or by tagging the site using 18O isotope labeling -LSB- 66 , 69 -RSB- . Using the HILIC method , 62 glycosylation sites could be identified for 37 glycoproteins in human plasma -LSB- 14 -RSB- , while a further study of the Cohn IV fraction of human plasma revealed 103 N-glycosylation sites and 23 fucosylated N-glycosylation sites -LSB- 66 -RSB- . 1465 N-glycosylated sites were found on 829 proteins in C. elegans -LSB- 72 -RSB- . 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 -LSB- 72 -RSB- , unlike the Cohn IV work , where a 1 mg fraction was used -LSB- 66 -RSB- . 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 -LSB- 65 , 70 -RSB- or the separation -LSB- 73 -RSB- 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 -LSB- 64 -RSB- or even the separation -LSB- 74 -RSB- 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 -LSB- 75 -RSB- . 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 -LSB- 76 -RSB- . The analysis of histone modifications on intact histone proteins by MS is very demanding , due to the enormous heterogeneity of the modifications -LSB- 77 -RSB- . 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 -LSB- 5 , 7 , 21 , 78 -- 80 -RSB- . 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 -LSB- 5 , 6 , 79 -- 81 -RSB- . 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 -LSB- 77 , 82 -RSB- . 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 -LSB- 82 -RSB- . 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 -LSB- 77 -RSB- . 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 -LRB- the three bigger peaks for the + butyrate sample -RRB- , and within these peaks a secondary separation is observed , relating to the number of methylations -LRB- the narrower peaks -RRB- . Adapted by permission from Macmillan Publishers Ltd : Nature Methods -LSB- 77 -RSB- 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 -LRB- the most commonly used peptide separation method -RRB- 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 .

O O O O O B O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O

post-translational modification
two-dimensional liquid chromatography


Improvement

of

the

liquid-chromatographic

analysis

of

protein

tryptic

digests

by

the

use

of

long-capillary

monolithic

columns

with

UV

and

MS

detection

Optimisation

of

peak

capacity

is

an

important

strategy

in

gradient

liquid

chromatography

-LRB-

LC

-RRB-

This

can

be

achieved

by

using

either

long

columns

or

columns

packed

with

small

particles

Monolithic

columns

allow

the

use

of

long

columns

at

relatively

low

back-pressure

The

gain

in

peak

capacity

using

long

columns

was

evaluated

by

the

separation

of

a

tryptic

bovine

serum

albumin

digest

with

an

LC

UV

mass

spectrometry

-LRB-

MS

-RRB-

system

and

monolithic

columns

of

different

length

-LRB-

and

mm

-RRB-

Peak

capacities

were

determined

from

UV

chromatograms

and

MS/MS

data

were

used

for

Mascot

database

searching

Analyses

with

a

similar

gradient

slope

for

the

two

columns

produced

ratios

of

the

peak

capacities

that

were

close

to

the

expected

value

of

the

square

root

of

the

column

length

ratio

Peak

capacities

of

the

short

column

were

and

with

and

min

gradients

respectively

and

and

for

the

long

column

with

and

min

gradients

respectively

Protein

identification

scores

were

also

higher

for

the

long

column

and

for

the

and

gradients

with

the

short

column

and

and

for

the

and

gradients

with

the

long

column

Thus

the

use

of

long

monolithic

columns

provides

improved

peptide

separation

and

increased

reliability

of

protein

identification

Introduction

Identification

of

unknown

proteins

is

a

key

step

in

proteome

analysis

The

standard

method

of

protein

identification

consists

of

enzymatic

digestion

of

the

protein

-LRB-

s

-RRB-

usually

by

using

trypsin

followed

by

mass

spectrometry

-LRB-

MS

-RRB-

analysis

of

the

resulting

digest

When

analysing

a

single

protein

e.g.

from

an

excised

gel

spot

a

peptide

separation

is

usually

unnecessary

When

analysing

a

digest

of

a

mixture

of

proteins

the

resolution

of

only

a

mass

spectrometer

is

usually

not

sufficient

In

those

cases

a

peptide

separation

is

applied

before

MS

detection

In

shotgun

proteomics

where

a

whole

proteome

is

digested

without

prior

protein

fractionation

liquid

chromatography

-LRB-

LC

-RRB-

is

the

method

of

choice

owing

to

the

high

complexity

of

the

peptide

mixtures

A

drawback

of

multidimensional

methods

is

the

time

frame

as

typical

analysis

times

for

analysis

of

peptide

mixtures

range

from

several

hours

to

more

than

day

-LSB-

-RSB-

methods

can

be

used

for

separation

of

less

complex

samples

-LRB-

digests

of

fewer

than

proteins

-RRB-

but

this

requires

very

efficient

separations

in

combination

with

the

additional

separation

power

of

MS

detection

Such

high-efficiency

separations

have

been

reported

for

analyses

with

long

packed

columns

using

-LRB-

ultra

-RRB-

high

pressure

-LRB-

about

MPa

-RRB-

systems

-LSB-

-RSB-

Peak

capacity

is

the

primary

parameter

for

evaluation

of

efficiency

in

gradient

chromatography

-LSB-

-RSB-

Peak

capacity

was

first

defined

by

Giddings

-LSB-

-RSB-

as

the

maximum

number

of

peaks

that

can

be

separated

by

a

phase

system

This

theory

was

later

adapted

for

gradient

chromatography

by

Horváth

and

Lipsky

-LSB-

-RSB-

There

are

generally

two

approaches

for

optimisation

of

the

peak

capacity

The

first

is

increase

of

the

gradient

length

gradients

of

up

to

h

have

been

reported

on

single

columns

-LSB-

-RSB-

However

according

to

theory

peak

capacity

increases

to

a

maximum

and

then

decreases

as

the

gradients

become

longer

-LSB-

-RSB-

The

second

approach

is

the

use

of

longer

columns

as

the

peak

capacity

increases

linearly

with

the

square

root

of

the

plate

number

and

thus

with

the

square

root

of

the

column

length

Wang

et

al.

-LSB-

-RSB-

illustrated

this

by

connecting

several

columns

packed

with

a

pellicular

stationary

phase

They

showed

that

the

ratio

of

the

peak

capacity

and

the

square

root

of

the

column

length

was

constant

for

columns

ranging

in

length

from

to

cm

The

main

limitation

to

simply

increasing

column

length

is

the

higher

back-pressure

A

possible

solution

for

this

problem

is

the

use

of

monolithic

columns

Monolithic

columns

have

a

higher

permeability

compared

with

packed

columns

facilitating

fast

separations

or

the

use

of

long

columns

-LSB-

-RSB-

Wang

et

al.

-LSB-

-RSB-

reported

a

back-pressure

of

MPa

for

a

cm

mm

packed

column

at

a

linear

flow

of

mm/s

In

contrast

other

groups

have

obtained

times

higher

linear

flow

rates

for

monolithic

columns

of

similar

length

-LSB-

-RSB-

The

higher

permeability

of

monolithic

columns

makes

it

possible

to

operate

long

columns

at

higher

flow

rates

while

using

conventional

LC

equipment

Luo

et

al.

-LSB-

-RSB-

separated

a

bacterial

protein

digest

at

a

linear

flow

rate

of

mm/s

with

a

monolithic

column

at

MPa

-LRB-

psi

-RRB-

Tolstikov

et

al.

-LSB-

-RSB-

analysed

plant

metabolomic

extracts

with

a

flow

rate

of

mm/s

for

a

column

The

quality

of

the

analysis

of

a

protein

digest

can

be

expressed

in

different

ways

One

way

is

to

describe

chromatographic

efficiency

in

terms

of

parameters

like

peak

width

peak

capacity

and

resolution

Another

way

is

to

use

the

reliability

of

protein

identification

like

SEQUEST

-LSB-

-RSB-

or

Mascot

-LRB-

-RRB-

-LSB-

-RSB-

scores

Such

an

approach

is

also

useful

as

the

mass

spectrometer

adds

a

second

dimension

to

the

separation

which

is

overlooked

when

only

chromatographic

efficiency

is

measured

In

this

paper

the

evaluation

of

capillary

monolithic

silica

columns

of

different

lengths

for

the

LC-UV-MS

analysis

of

a

bovine

serum

albumin

-LRB-

BSA

-RRB-

tryptic

digest

is

described

Columns

of

and

length

were

investigated

using

gradient

times

varying

from

to

min

Chromatographic

peak

capacities

based

on

UV

detection

and

protein

identification

based

on

Mascot

scoring

data

of

the

MS

detection

were

determined

as

a

measure

for

the

efficiency

of

peptide

separation

Theoretical

aspects

The

peak

capacity

is

defined

as

the

maximum

number

of

bands

that

will

fit

within

a

chromatogram

with

a

resolution

of

Rs

-LSB-

-RSB-

In

gradient

LC

where

peak

width

is

about

constant

throughout

the

separation

the

theoretical

peak

capacity

is

given

by

Eq

where

tg

is

the

gradient

time

and

wav

is

the

average

baseline

peak

width

For

large

values

of

PC

this

approaches

tg/wav

Because

sample

peaks

often

do

not

occupy

the

entire

length

of

the

gradient

the

sample

peak

capacity

can

then

be

defined

as

where

ta

and

tz

are

the

retention

times

of

the

first

and

last

eluting

peaks

respectively

Wang

et

al.

-LSB-

-RSB-

investigated

the

effect

of

column

length

on

peak

capacity

in

packed

columns

and

found

that

the

peak

capacity

is

proportional

to

if

the

gradient

slope

is

proportional

to

L

The

gradient

slope

can

be

defined

as

where

Δϕ

is

the

change

in

organic

modifier

fraction

during

the

gradient

-LRB-

ϕ

-RRB-

and

is

the

column

dead

time

If

the

linear

flow

rate

is

constant

is

only

dependent

on

column

length

therefore

tg

should

be

varied

proportionally

to

column

length

in

order

to

keep

the

gradient

slope

constant

Experimental

Materials

and

reagents

BSA

trypsin

-LRB-

porcine

type

IX-S

EC

-RRB-

and

-LRB-

DTT

-RRB-

were

purchased

from

Sigma

-LRB-

St.

Louis

MO

USA

-RRB-

and

iodoacetamide

-LRB-

IAA

-RRB-

and

from

Fluka

-LRB-

Buchs

Switzerland

-RRB-

Acetonitrile

was

high-performance

LC

gradient

grade

-LRB-

Biosolve

Valkenswaard

the

Netherlands

-RRB-

and

spectroscopy-grade

trifluoroacetic

acid

-LRB-

TFA

-RRB-

was

obtained

from

Merck

-LRB-

Darmstadt

Germany

-RRB-

All

solutions

were

prepared

using

water

from

a

Milli-Q

water-purifying

system

-LRB-

Millipore

Bedford

MA

USA

-RRB-

All

reagents

for

the

digestion

of

BSA

were

prepared

in

mM

buffer

pH

The

tryptic

digest

was

prepared

as

follows

A

aliquot

of

BSA

stock

solution

-LRB-

μg/μl

in

water

-RRB-

was

set

to

pH

by

adding

μl

of

M

buffer

-LRB-

pH

-RRB-

After

addition

of

μl

of

mM

DTT

solution

the

sample

was

incubated

at

°C

for

min

to

reduce

disulfide

bonds

After

cooling

to

room

temperature

μl

of

mM

IAA

solution

was

added

and

the

sample

was

incubated

in

the

dark

for

min

to

alkylate

the

free

thiols

Trypsin

was

dissolved

in

μl

of

buffer

to

reach

a

trypsin-to-protein

mass

ratio

of

in

the

final

solution

the

trypsin

solution

was

added

to

the

sample

which

was

incubated

at

°C

overnight

for

h

The

digestion

was

stopped

by

addition

of

μl

of

TFA

The

sample

was

diluted

to

ng/μl

-LRB-

μM

-RRB-

with

LC

mobile

phase

A

-LRB-

water

plus

TFA

-RRB-

and

injected

without

further

purification

Apparatus

and

LC

columns

All

analyses

were

performed

with

an

Agilent

nanoLC

system

-LRB-

Agilent

Technologies

Waldbronn

Germany

-RRB-

consisting

of

a

vacuum

degasser

a

binary

Nano-Pump

a

μ-well

plate

sampler

and

a

column

switching

module

with

a

trapping

column

in

the

position

of

the

six-port

column-switching

valve

The

trapping

pump

was

a

Gynkotek

model

-LRB-

Gynkotek

Germering

Germany

-RRB-

Detection

was

performed

by

UV

and

MS

detection

with

the

detectors

connected

in

series

The

UV

detector

was

an

MU

UV

VIS

detector

-LRB-

ATAS

GL

International

Veldhoven

the

Netherlands

-RRB-

equipped

with

an

external

optical-fibre

flow

cell

-LRB-

nl

light

path

-RRB-

peptides

were

detected

at

nm

The

mass

spectrometer

was

an

Agilent

LC/MSD

Trap

XCT

-LRB-

Agilent

Technologies

Waldbronn

Germany

-RRB-

ion-trap

mass

spectrometer

equipped

with

an

orthogonal

electrospray

ionisation

-LRB-

ESI

-RRB-

interface

The

external

flow

cell

of

the

UV

detector

allows

minimal

time

delay

and

band-broadening

between

UV

and

MS

detection

The

monolithic

columns

were

provided

by

GL

Sciences

-LRB-

Tokyo

Japan

-RRB-

The

columns

were

a

mm

mm

MonoCap

for

nano-flow

monolithic

column

and

a

mm

mm

MonoCap

high

resolution

monolithic

column

For

trapping

of

the

digest

a

mm

mm

column

packed

with

μm

Zorbax

-LRB-

Agilent

Technologies

Waldbronn

Germany

-RRB-

was

used

Method

and

data

analysis

LC

solvent

A

was

water

plus

TFA

-LRB-

v/v

-RRB-

solvent

B

was

acetonitrile

plus

TFA

-LRB-

v/v

-RRB-

The

trapping

solvent

was

a

mixture

of

-LRB-

v/v

-RRB-

solvent

B

in

solvent

A

After

injection

of

the

digest

-LRB-

μl

for

the

inner

diameter

column

and

μl

for

the

inner

diameter

column

-RRB-

the

sample

was

trapped

on

the

trapping

column

at

a

flow

rate

of

μl/min

After

min

the

trapping

column

was

switched

on-line

with

the

separation

column

and

the

gradient

was

started

All

separations

were

performed

at

room

temperature

using

a

gradient

of

solvent

B

with

gradient

times

varying

from

to

min

MS

spectra

were

acquired

in

the

positive

ion

mode

over

the

m/z

range

after

which

the

two

most

intense

ions

-LRB-

with

a

preference

for

doubly

charged

ions

-RRB-

were

selected

for

fragmentation

MS/MS

fragmentation

spectra

were

acquired

over

the

m/z

range

An

ESI

spray

voltage

of

kV

was

used

for

all

experiments

The

effect

of

separation

efficiency

on

protein

identification

was

evaluated

using

the

Mascot

search

engine

-LSB-

-RSB-

LC-MS/MS

data

were

converted

to

the

Mascot

generic

format

-LRB-

mgf

file

-RRB-

using

the

data-analysis

software

and

the

mgf

files

were

searched

against

the

MSDB

database

using

Mascot

's

MS/MS

ion

search

module

The

database

was

searched

for

tryptic

peptides

from

all

entries

in

the

database

allowing

one

missed

cleavage

per

peptide

and

containing

carbamidomethyl

cysteine

as

a

variable

modification

Mass

tolerances

were

set

to

default

values

peptide

mass

tolerance

Da

MS/MS

tolerance

Da

Results

and

discussion

Liquid

chromatography

UV

analysis

Because

of

the

difference

in

diameter

the

mm

mm

and

the

mm

mm

columns

were

used

with

different

flow

rates

For

the

and

the

columns

the

flow

rates

were

and

μl/min

respectively

resulting

in

a

linear

flow

rate

of

mm/s

Injection

volumes

were

also

proportional

to

the

square

of

the

column

diameter

μl

of

the

digest

for

the

column

and

μl

onto

the

column

During

the

gradient

the

maximum

back-pressure

of

the

column

was

below

Mpa

which

is

well

below

the

manufacturers

limit

of

Mpa

Figure

shows

the

LC-UV

chromatograms

of

and

gradients

run

on

the

column

and

and

gradients

run

on

the

column

When

the

chromatograms

of

the

analyses

with

similar

gradient

slopes

are

compared

-LRB-

Fig.

b

and

Fig.

d

-RRB-

it

is

clear

that

an

increase

in

column

length

improves

the

peptide

separation

In

order

to

quantify

the

efficiency

of

the

separation

the

sample

peak

capacity

was

calculated

for

all

analyses

Because

of

the

incomplete

resolution

of

the

digest

the

peak

capacity

was

estimated

by

using

the

average

peak

width

of

a

selected

number

of

peaks

that

appeared

to

contain

only

a

single

peptide

Using

this

method

we

calculated

peak

capacities

for

all

analyses

and

the

results

are

summarised

in

Table

The

peak

capacities

found

for

the

short

column

are

comparable

to

those

found

in

the

literature

for

similar

columns

-LSB-

-RSB-

As

expected

the

peak

capacities

of

the

long

column

are

higher

than

those

of

the

short

column

but

they

are

relatively

low

compared

with

the

values

reported

in

-LSB-

-RSB-

However

when

gradient

time

is

taken

into

consideration

the

difference

is

significantly

less

PC

tg

is

peaks

per

minute

with

the

present

system

-LRB-

gradient

on

the

column

-RRB-

and

peaks

per

minute

for

the

gradient

reported

in

-LSB-

-RSB-

A

possible

negative

effect

on

the

resolution

of

our

system

is

the

use

of

a

trapping

column

filled

with

a

different

stationary

phase

which

probably

has

a

selectivity

that

differs

from

that

of

the

analysis

column

Fig.

chromatography

-LRB-

LC

-RRB-

UV

chromatograms

of

a

tryptic

bovine

serum

albumin

-LRB-

BSA

-RRB-

digest

separated

by

monolithic

silica

capillary

columns

of

mm

mm

-LRB-

a

c

-RRB-

and

mm

mm

-LRB-

b

d

-RRB-

using

a

gradient

of

acetonitrile

-LRB-

v/v

trifluoroacetic

acid

TFA

-RRB-

in

water

-LRB-

v/v

TFA

-RRB-

a

A

gradient

-LRB-

min

-RRB-

b

c

gradient

-LRB-

min

-RRB-

d

gradient

-LRB-

min

-RRB-

The

run

times

include

min

of

trapping

and

and

min

of

gradient

delay

time

for

the

and

columns

respectivelyTable

parameters

from

liquid

chromatography

UV

analysis

mm

mm

mmtg

-LRB-

min

-RRB-

wavΔtPC

.243.0512.60.283.1611.2150.4411.025.00.3911.629.7751.2737.629.60.7731.741.0

tg

gradient

time

wav

average

width

of

seven

to

peaks

which

were

found

to

contain

only

a

single

peptide

-LRB-

based

on

mass

spectrometry

data

-RRB-

Δt

elution

window

between

the

first

and

last

eluting

peptides

PC

sample

peak

capacity

-LRB-

Δt/wav

-RRB-

If

chromatograms

with

the

same

gradient

slope

are

compared

-LRB-

and

gradients

for

the

column

and

and

gradients

for

the

column

respectively

-RRB-

the

ratio

of

the

peak

capacities

should

be

close

to

the

square

root

of

the

column

length

ratio

-LRB-

-RRB-

For

the

gradient

pair

this

ratio

is

for

the

gradient

pair

the

ratio

is

For

the

short

column

no

increase

in

peak

capacity

is

observed

with

a

gradient

of

more

than

min

but

for

the

long

column

the

peak

capacity

increases

up

to

a

gradient

A

possible

explanation

can

be

found

in

the

study

of

Stadalius

et

al.

-LSB-

-RSB-

who

demonstrated

that

peak

capacity

will

increase

with

gradient

time

until

it

reaches

a

maximum

after

which

it

will

even

decrease

The

gradient

time

at

which

this

maximum

is

obtained

is

greater

for

longer

columns

Liquid

chromatography

mass

spectrometry

analysis

To

assess

the

influence

of

peptide

separation

on

protein

identification

MS/MS

data

were

investigated

by

database-searching

using

the

MS/MS

ion

search

module

from

the

Mascot

search

engine

The

results

are

expressed

as

a

Mascot

score

the

number

of

unique

identified

peptides

and

the

percentage

of

the

BSA

amino

acid

sequence

covered

by

these

peptides

-LRB-

Table

-RRB-

These

results

were

compared

with

the

scores

obtained

for

direct

infusion

of

the

BSA

digest

at

the

same

flow

rates

as

for

the

LC

separations

The

protein

identification

parameters

for

the

infusion

experiments

were

about

similar

for

both

flow

rates

All

database

searches

gave

bovine

albumin

as

the

top

protein

match

and

the

only

other

significant

matches

were

albumins

from

other

species

Table

database

search

results

mm

mm

mmtgMascot

scorePeptidesaCoverageb

-LRB-

-RRB-

Mascot

scorePeptidesaCoverageb

-LRB-

-RRB-

of

unique

peptides

identified

by

database

search.bPercentage

of

amino

acid

sequence

covered

by

identified

peptides.cDirect

infusion

of

the

digest

into

the

mass

spectrometer

Figure

shows

base

peak

chromatograms

of

separations

with

the

same

gradient

slope

a

gradient

for

the

column

and

a

gradient

for

the

column

The

Mascot

scores

for

the

column

are

higher

than

those

for

the

column

The

average

score

per

identified

peptide

is

between

and

for

the

long

column

and

between

and

for

the

short

column

Combined

with

the

larger

number

of

identified

peptides

on

the

long

column

this

adds

up

to

a

higher

Mascot

score

-LRB-

Table

-RRB-

Fig.

mass

spectrometry

-LRB-

MS

-RRB-

base

peak

chromatograms

and

mass

spectra

of

tryptic

BSA

digest

separated

by

monolithic

silica

columns

of

mm

mm

-LRB-

a

-RRB-

and

mm

mm

-LRB-

b

-RRB-

using

a

gradient

of

acetonitrile

-LRB-

v/v

TFA

-RRB-

in

water

-LRB-

v/v

TFA

-RRB-

a

A

gradient

-LRB-

min

-RRB-

b

gradient

-LRB-

min

-RRB-

The

run

times

include

min

of

trapping

and

and

min

of

gradient

delay

time

for

the

and

columns

respectively

Despite

providing

only

a

low

resolution

even

the

shortest

gradients

cause

a

considerable

increase

in

the

reliability

of

protein

identification

Increase

in

gradient

length

leads

to

cleaner

mass

spectra

-LRB-

Fig.

-RRB-

and

a

higher

number

of

identified

peptides

and

sequence

coverage

compared

with

direct

infusion

These

numbers

however

decrease

beyond

a

certain

gradient

time

This

could

possibly

be

attributed

to

the

lower

peak

heights

for

longer

gradient

times

This

result

indicates

that

there

is

a

gradient

slope

where

an

increase

in

chromatographic

separation

no

longer

improves

protein

identification

Fig.

mass

spectra

of

peptide

YICDNQDTISSK

-LRB-

m/z

-RRB-

as

identified

from

extracted

ion

chromatograms

in

the

LC-MS

analysis

of

a

tryptic

BSA

digest

a

A

silica

monolithic

column

gradient

of

acetonitrile

-LRB-

v/v

TFA

-RRB-

in

water

-LRB-

v/v

TFA

-RRB-

b

column

gradient

Conclusions

The

use

of

long

silica-based

capillary

monolithic

columns

provides

a

clear

advantage

over

use

of

shorter

columns

i.e.

an

increase

of

chromatographic

efficiency

and

reliability

of

protein

identification

As

expected

from

chromatography

theory

a

factor

longer

column

gives

a

times

increase

in

peak

capacity

for

separations

with

similar

gradient

slope

The

use

of

longer

gradients

also

leads

to

an

initial

improvement

of

the

protein

identification

score

but

the

score

seems

to

have

a

maximum

at

longer

gradient

times

While

the

use

of

longer

columns

for

the

separation

of

peptides

has

a

clear

advantage

because

of

the

gain

in

chromatographic

efficiency

this

also

gives

a

longer

analysis

time

As

maximum

protein

identification

scores

for

rather

simple

digests

are

reached

at

relatively

short

gradient

times

it

is

important

to

find

a

compromise

between

chromatographic

efficiency

and

analysis

time

However

if

the

sample

is

more

complex

the

use

of

longer

columns

is

more

attractive

as

longer

gradients

are

necessary

to

achieve

sufficient

separation

In

the

near

future

short

and

long

columns

of

the

same

diameter

-LRB-

inner

diameter

-RRB-

will

be

compared

Further

improvement

of

the

separation

might

be

obtained

by

optimisation

of

the

combination

of

the

trap

column

and

the

analysis

column

Moreover

the

potential

of

longer

monolithic

capillary

columns

will

be

demonstrated

by

the

analysis

of

more

complex

and

real

samples

O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I I O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

peak capacity
long columns
gradient liquid chromatography
protein identification