Patent Description:
Sialylated glycoconjugates are known to play key roles in several pathophysiological processes including viral infection, embryogenesis, inflammation, cardiovascular diseases, cancer, and neural development. The most common mammalian sialic acids comprise different linkages of N-acetylneuraminic acid (Neu5Ac). These linkages include Neu5Ac-alpha (<NUM>,<NUM>) galactose (Gal) (hereinafter "a2,<NUM>") and Neu5Ac-alpha (<NUM>,<NUM>) Gal (hereinafter "a2,<NUM>"). Determining the relative abundances of α2,<NUM> and a2,<NUM> are important in diagnosing the pathophysiological processes just described.

The stereochemistry structural identification in N-glycans or N-glycopeptides of a2,<NUM> and a2,<NUM> is conventionally reported by separation techniques such as capillary electrophoresis (CE) and ion mobility (IMS). Unfortunately, no methods are reported using liquid chromatography coupled mass spectrometry (LC-MS) of non-derivatized glycopeptides by conventional proteomics shotgun sample preparation. The use of LC-MS and shotgun sample preparation (non-derivatized glycopeptides) reduces experimental complexity and increases overall sample throughput. Instead, biochemical identifications employ linkage specific sialic acid derivatization of the glycan before MS. In other words, these techniques generally require chemical conversion of the glycan to a derivative before mass spectrometry (MS).

For example, "High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification" by Reiding et al. (hereinafter the "Reiding Paper") describes a method for sialic acid stabilization and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to study glycosylation. The Reiding Paper employs a combination of carboxylic acid activators in ethanol to achieve near-complete ethyl esterification of a2,<NUM> sialic acids and lactonization of a2,<NUM> variants. In other words, the Reiding Paper describes an ethyl esterification chemical conversion of the glycan before MS.

Similarly, "Collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides" by Zhao et al. (hereinafter the "Zhao Paper") describes a method to confirm the sequence, branching, and linkage assignments for a glycan. The Zhao Paper is not directed to determining α2,<NUM> and a2,<NUM> linkages. However, the Zhao Paper does describe subjecting glycans to permethylation to increase their sensitivity to collisionally-activated dissociation (CAD) and electron capture dissociation (ECD). In other words, the Zhao Paper describes a permethylation chemical conversion of the glycan before MS. For these glycans, CAD and "hot" ECD provide complementary structural information.

As a result, systems and methods are needed to determine abundances of sialic acid linkages α2,<NUM> and a2,<NUM> of glycopeptides using LC-MS or LC-MS/MS of non-derivatized glycopeptides by conventional proteomics shotgun sample preparation.

Mass spectrometers are often coupled with chromatography or other separation systems, such as ion mobility, to identify and characterize eluting known compounds of interest from a sample. In such a coupled system, the eluting solvent is ionized and a series of mass spectra are obtained from the eluting solvent at specified time intervals called retention times. These retention times range from, for example, <NUM> second to <NUM> minutes or greater. The series of intensities of an ion of mass spectra measured at the retention times form a chromatogram, or extracted ion chromatogram (XIC).

Peaks found in the XIC are used to identify or characterize a known peptide or compound in the sample. More particularly, the retention times of peaks and/or the area of peaks are used to identify or characterize (quantify) a known peptide or compound in the sample.

In traditional separation coupled mass spectrometry systems, a fragment or product ion of a known compound is selected for analysis. A tandem mass spectrometry or mass spectrometry/ mass spectrometry (MS/MS) scan is then performed at each interval of the separation for a mass range that includes the product ion. The intensity of the product ion found in each MS/MS scan is collected over time and analyzed as a collection of spectra, or an XIC, for example.

In general, tandem mass spectrometry, or MS/MS, is a well-known technique for analyzing compounds. Tandem mass spectrometry involves ionization of one or more compounds from a sample, selection of one or more precursor ions of the one or more compounds, fragmentation of the one or more precursor ions into fragment or product ions, and mass analysis of the product ions.

Tandem mass spectrometry can provide both qualitative and quantitative information. The product ion spectrum can be used to identify a molecule of interest. The intensity of one or more product ions can be used to quantitate the amount of the compound present in a sample.

A large number of different types of experimental methods or workflows can be performed using a tandem mass spectrometer. Three broad categories of these workflows are targeted acquisition, information dependent acquisition (IDA) or data-dependent acquisition (DDA), and data-independent acquisition (DIA).

In a targeted acquisition method, one or more transitions of a precursor ion to a product ion are predefined for a compound of interest. As a sample is being introduced into the tandem mass spectrometer, the one or more transitions are interrogated or monitored during each time period or cycle of a plurality of time periods or cycles. In other words, the mass spectrometer selects and fragments the precursor ion of each transition and performs a targeted mass analysis only for the product ion of the transition. As a result, an intensity (a product ion intensity) is produced for each transition. Targeted acquisition methods include, but are not limited to, multiple reaction monitoring (MRM) and selected reaction monitoring (SRM).

Multiple Reaction Monitoring (MRM) on triple quadrupole based instrumentation is the standard mass spectrometric technique of choice for targeted MS quantification in all application areas, due to its ability to provide the highest specificity and sensitivity for the detection of specific components in complex mixtures. However, the speed and sensitivity of today's accurate mass MS systems have enabled a new quantification strategy with similar performance characteristics. In this strategy (termed MRM-HR workflow or parallel reaction monitoring, PRM), looped MS/MS spectra are collected at high-resolution with short accumulation times, and then fragment ions are extracted post-acquisition to generate MRM-like peaks for integration and quantification. With instrumentation like the TRIPLETOF® Systems, this targeted technique is sensitive and fast enough to enable quantitative performance similar to higher end triple quadrupole instruments, with full fragmentation data measured at high resolution and high mass accuracy.

In an IDA method, a user can specify criteria for performing an untargeted mass analysis of product ions, while a sample is being introduced into the tandem mass spectrometer. For example, in an IDA method, a precursor ion or mass spectrometry (MS) survey scan is performed to generate a precursor ion peak list. The user can select criteria to filter the peak list for a subset of the precursor ions on the peak list. MS/MS is then performed on each precursor ion of the subset of precursor ions. A product ion spectrum is produced for each precursor ion. MS/MS is repeatedly performed on the precursor ions of the subset of precursor ions as the sample is being introduced into the tandem mass spectrometer.

In proteomics and many other sample types, however, the complexity and dynamic range of compounds are very large. This poses challenges for traditional targeted and IDA methods, requiring very high-speed MS/MS acquisition to deeply interrogate the sample in order to both identify and quantify a broad range of analytes.

As a result, DIA methods, the third broad category of tandem mass spectrometry, were developed. These DIA methods have been used to increase the reproducibility and comprehensiveness of data collection from complex samples. DIA methods can also be called non-specific fragmentation methods. In a traditional DIA method, the actions of the tandem mass spectrometer are not varied among MS/MS scans based on data acquired in a previous precursor or product ion scan. Instead, a precursor ion mass range is selected. A precursor ion mass selection window is then stepped across the precursor ion mass range. All precursor ions in the precursor ion mass selection window are fragmented and all of the product ions of all of the precursor ions in the precursor ion mass selection window are mass analyzed.

The precursor ion mass selection window used to scan the mass range can be very narrow so that the likelihood of multiple precursors within the window is small. This type of DIA method is called, for example, MS/MSALL. In an MS/MSALL method, a precursor ion mass selection window of about <NUM> amu is scanned or stepped across an entire mass range. A product ion spectrum is produced for each <NUM> amu precursor mass window. The time it takes to analyze or scan the entire mass range once is referred to as one scan cycle. Scanning a narrow precursor ion mass selection window across a wide precursor ion mass range during each cycle, however, is not practical for some instruments and experiments.

As a result, a larger precursor ion mass selection window, or selection window with a greater width, is stepped across the entire precursor mass range. This type of DIA method is called, for example, SWATH acquisition. In a SWATH acquisition, the precursor ion mass selection window stepped across the precursor mass range in each cycle may have a width of <NUM>-<NUM> amu, or even larger. Like the MS/MSALL method, all the precursor ions in each precursor ion mass selection window are fragmented, and all of the product ions of all of the precursor ions in each mass selection window are mass analyzed.

Electron-based dissociation (ExD), ultraviolet photodissociation (UVPD), infrared photodissociation (IRMPD) and collision-induced dissociation (CID) or CAD are often used as fragmentation techniques for tandem mass spectrometry (MS/MS). ExD can include, but is not limited to, ECD or electron transfer dissociation (ETD). CID is the most conventional technique for dissociation in tandem mass spectrometers.

As described above, in top-down and middle-down proteomics, an intact or digested protein is ionized and subjected to tandem mass spectrometry. ECD, for example, is a dissociation technique that dissociates peptide and protein backbones preferentially. As a result, this technique is an ideal tool to analyze peptide or protein sequences using a top-down and middle-down proteomics approach.

The present invention generally relates to a system, a method, and a computer program product for identifying one or more linkages of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, in accordance with various embodiments. The system includes a separation device, an ion source, a tandem mass spectrometer, and a processor.

The system of the present invention is defined in claim <NUM>.

The separation device separates one or more isomers of a glycopeptide from a sample. The ion source ionizes the one or more separated isomers, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide.

The tandem mass spectrometer includes an ECD device. For each separation time of a plurality separation times, the tandem mass spectrometer executes or monitors a first and a second group of MRM transitions using the ECD device with an electron energy of <NUM>-<NUM> eV. The first group of one or more MRM transitions is selected so that each transition includes the precursor ion and a product ion known to be enhanced or suppressed for a first linkage of a sialic acid of the glycopeptide to a glycan. The second group of one or more MRM transitions is selected so that each transition includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage of the sialic acid of the glycopeptide to the glycan. In various embodiments, MRM-HR can be used to monitor all product ions simultaneously. An XIC is produced for the precursor ion. An XIC is produced for each product ion of the first and second groups.

The processor performs a number of steps. The processor calculates a separation time of an isomer of the one or more separated isomers from a peak of the TIC. The processor sums product ion intensities of the first group at the separation time producing a first sum and sums product ion intensities of the second group at the separation time producing a second sum using XICs of the first and second groups. The processor calculates a ratio of the first sum to the second sum. The processor compares the ratio at the separation time to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times. The one or more times correspond to the number of one or more sialic acids known to be included in the glycopeptide. Finally, the processor identifies one or more linkages of the sialic acid to the glycan of the isomer from the combination found to match the ratio in the comparison.

These and other features of the applicant's teachings are set forth herein.

Before one or more embodiments of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings.

<FIG> is a block diagram that illustrates a computer system <NUM>, upon which embodiments of the present teachings may be implemented. Computer system <NUM> includes a bus <NUM> or other communication mechanism for communicating information, and a processor <NUM> coupled with bus <NUM> for processing information. Computer system <NUM> also includes a memory <NUM>, which can be a random-access memory (RAM) or other dynamic storage device, coupled to bus <NUM> for storing instructions to be executed by processor <NUM>. Memory <NUM> also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor <NUM>.

Computer system <NUM> may be coupled via bus <NUM> to a display <NUM>, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. Another type of user input device is cursor control <NUM>, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor <NUM> and for controlling cursor movement on display <NUM>. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

A computer system <NUM> can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system <NUM> in response to processor <NUM> executing one or more sequences of one or more instructions contained in memory <NUM>. Such instructions may be read into memory <NUM> from another computer-readable medium, such as storage device <NUM>. Execution of the sequences of instructions contained in memory <NUM> causes processor <NUM> to perform the process described herein. Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

In various embodiments, computer system <NUM> can be connected to one or more other computer systems, like computer system <NUM>, across a network to form a networked system. The network can include a private network or a public network such as the Internet. In the networked system, one or more computer systems can store and serve the data to other computer systems. The one or more computer systems that store and serve the data can be referred to as servers or the cloud, in a cloud computing scenario. The one or more computer systems can include one or more web servers, for example. The other computer systems that send and receive data to and from the servers or the cloud can be referred to as client or cloud devices, for example.

The term "computer-readable medium" as used herein refers to any media that participates in providing instructions to processor <NUM> for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Volatile media includes dynamic memory, such as memory <NUM>. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus <NUM>.

Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor <NUM> for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. An infra-red detector coupled to bus <NUM> can receive the data carried in the infra-red signal and place the data on bus <NUM>. Bus <NUM> carries the data to memory <NUM>, from which processor <NUM> retrieves and executes the instructions. The instructions received by memory <NUM> may optionally be stored on storage device <NUM> either before or after execution by processor <NUM>.

The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description.

The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

As described above, determining the relative abundances of α2,<NUM> and α2,<NUM> are important in diagnosing pathophysiological processes including, but not limited to, viral infection, embryogenesis, inflammation, cardiovascular diseases, cancer, and neural development. The stereochemistry structural identification in N-glycans or N-glycopeptides of a2,<NUM> and α2,<NUM> is conventionally reported by separation techniques such as CE and IMS. Unfortunately, no methods are reported using LC-MS or LC-MS/MS of non-derivatized glycopeptides by conventional proteomics shotgun sample preparation. The use of LC-MS and shotgun sample preparation (non-derivatized glycopeptides) reduces experimental complexity and increases overall sample throughput.

In various embodiments, isomers of a glycopeptide with different combinations of linkages a2,<NUM> and α2,<NUM> for the sialic acids of the glycopeptide are identified using LC-MS/MS. The isomers are separated using LC. Groups of MRM transitions targeting linkages a2,<NUM> and α2,<NUM> are monitored using ECD with an electron energy between <NUM>-<NUM> eV as the isomers are eluting. A ratio of the intensities measured from the two different groups of transitions is used to identify the sialic linkages of the isomers as a2,<NUM> or a2,<NUM>. The relative abundances or quantities of the isomers with different sialic linkages are then used to diagnose pathophysiological processes.

A glycopeptide, for example, is a peptide that includes a glycan. A glycan is, for example, a carbohydrate or complex sugar. Glycans are known to attach to cell surface receptor proteins or extracellular proteins such as antibodies and have a large variety of specific biological functions. A peptide backbone is a sequence of amino acids. If a glycan is attached to the amino acid asparagine (N) of a peptide backbone, the peptide is referred to as an N-glycopeptide.

<FIG> is an exemplary diagram <NUM> showing a structural formula and an Oxford system notation for a glycan that includes two sialic acids that each has a different sialic acid to sugar linkage, in accordance with various embodiments. In <FIG>, structural formula <NUM> shows a sialic acid, Neu5Ac <NUM>, connected at the end of one antenna of the glycan and another sialic acid, Neu5Ac <NUM>, connected at the end of another antenna of the glycan. Neu5Ac <NUM> is connected to a Gal of the glycan through a2,<NUM> linkage <NUM>. Neu5Ac <NUM> is connected to a Gal of the glycan through a2,<NUM> linkage <NUM>.

Equivalently, Oxford system notation <NUM> shows Neu5Ac <NUM> connected at the end of one antenna of the glycan and Neu5Ac <NUM> connected at the end of another antenna of the glycan. Neu5Ac <NUM> is connected to Gal <NUM> of the glycan through a2,<NUM> linkage <NUM>. Neu5Ac <NUM> is connected to Gal <NUM> of the glycan through a2,<NUM> linkage <NUM>. In Oxford system notation <NUM>, a2,<NUM> linkage <NUM> and a2,<NUM> linkage <NUM> are bent in different directions with respect to their antenna to show that they are different linkages. At the end of the glycan opposite Neu5Ac <NUM> and Neu5Ac <NUM>, the glycan is connected or attached to the backbone of a peptide (not shown), for example.

Other components of the glycan include Mannose (Man) <NUM> and N-Acetylglucosamine (GlcNAc) <NUM>. Gal <NUM>, Gal <NUM>, and Man <NUM> are types of hexose. GlcNAc <NUM> and N-Acetylgalactosamine (GalNAc) are types of hexNAc. In <FIG>, the sialic acids are shown as Neu5Ac <NUM> and Neu5Ac <NUM>. In various alternative embodiments, the sialic acids can be, but are not limited to, N-Glycolylneuraminic acid (Neu5Gc, or Sg). Humans, for example, do not have Sg.

In various embodiments, glycopeptides, such as N-glycopeptides are found from digested proteins and are separated by LC. For example, LC separation methods for glycopeptides include, but are not limited to, reversed-phase LC and non-reversed hydrophilic interaction liquid chromatography (HILIC).

<FIG> is an exemplary diagram <NUM> showing a glycan with one sialic acid attached to a peptide backbone forming a glycopeptide, in accordance with various embodiments. In <FIG>, glycan <NUM> is attached at one end to peptide backbone <NUM>. As described above, peptide backbone <NUM> is made up of a series of one or more amino acids. Glycan <NUM> includes two antennae <NUM> and <NUM>, two Gal sugars <NUM> and <NUM>, and one sialic acid Neu5Ac <NUM>. A glycopeptide with the configuration of glycan <NUM> is sometimes referred to as an A2G2Sa1 glycopeptide, referring to two antennae, two Gal sugars, and one sialic acid, respectively.

Glycan <NUM> has only one sialic acid Neu5Ac <NUM> and, therefore, represents the simplest case. In <FIG>, the linkage of Neu5Ac <NUM> to Gal sugars <NUM> and <NUM> is not shown as bent to the left or the right. As a result, it does not depict either of the two possible linkages a2,<NUM> or a2,<NUM>.

In various embodiments, in order to determine the linkage of Neu5Ac <NUM> to Gal sugars <NUM> and <NUM>, the glycopeptide of <FIG> is subjected to MS as it is eluting from the LC column. The m/z of the glycopeptide is <NUM>, for example. From the MS, a precursor XIC of the glycopeptide is obtained.

<FIG> is an exemplary plot <NUM> of a precursor XIC from MS of the glycopeptide of <FIG>, in accordance with various embodiments. Precursor XIC <NUM> includes two precursor ion peaks <NUM> and <NUM> for the glycopeptide of <FIG>. Peaks <NUM> and <NUM> in <FIG> represent isomers of the glycopeptide. In other words, they have the same m/z of <NUM> but a different structure. Specifically, peaks <NUM> and <NUM> represent different linkages of Neu5Ac <NUM> to Gal sugars <NUM> and <NUM> in <FIG>. In other words, returning to <FIG>, one of peaks <NUM> and <NUM> in <FIG> represent an a2,<NUM> linkage and the other peak represents an α2,<NUM> linkage.

Conventionally, as described above in reference to the Reiding Paper and the Zhao Paper, it was not thought possible to distinguish a2,<NUM> and a2,<NUM> linkages without employing linkage specific sialic acid derivatization of the glycan before MS. More broadly, it was known that ExD and CID are complementary for glycopeptide analysis. However, it was generally thought that ExD, or ECD specifically, works on the peptide backbone in glycopeptides and CID works on glycans in glycopeptides, although ECD has been shown to work on free glycans also. In general, it was known that "low" ECD (<NUM>-<NUM> eV) and ETD do not provide good dissociation efficiency and "hot" ECD (<NUM>-<NUM> eV) is better for more complex glycans. The Zhao Paper, for example, describes that hot ECD on a doubly charged disialyated glycan is not useful. In contrast, the Zhao Paper reports that hot ECD provides more structural information than CID for larger complex glycans.

In various embodiments, experimentation shows that ECD with an electron energy between <NUM> and <NUM> eV, preferably <NUM> eV, produces detectable fragments of glycans in glycopeptides. These fragments are then used to distinguish between a2,<NUM> and a2,<NUM> sialic acid linkages of the glycans.

Returning to <FIG>, two groups of fragment or product ions <NUM> and <NUM> for the glycan of the glycopeptide of <FIG> are shown. When this glycopeptide (<NUM>/z) is fragmented using ECD with an electron energy of <NUM> eV, group <NUM> product ions are found to be enhanced for a2,<NUM> sialic acid linkages and group <NUM> product ions are found to be suppressed for α2,<NUM> sialic acid linkages. Conversely, under the same conditions, group <NUM> product ions are found to be suppressed for a2,<NUM> sialic acid linkages and group <NUM> product ions are found to be enhanced for a2,<NUM> sialic acid linkages. In other words, using ECD with an electron energy of <NUM> eV, a2,<NUM> and α2,<NUM> sialic acid linkages can be identified by comparing the intensities of different groups of product ions.

<FIG> is an exemplary series <NUM> of plots of two XICs measured for two different groups of MRM transitions developed for the glycopeptide of <FIG> and monitored using ECD with an electron energy of <NUM> eV, in accordance with various embodiments. XIC <NUM> in plot <NUM> represents a sum of the intensities measured for three MRM transitions <NUM> to <NUM>, <NUM>, and <NUM>/z measured over a series of LC separation times. XIC <NUM> in plot <NUM> represents a sum of the intensities measured for three MRM transitions <NUM> to <NUM>, <NUM>, and <NUM>/z measured over a series of LC separation times.

Both XIC <NUM> and XIC <NUM> produce product ion peaks at times <NUM> and <NUM> corresponding to precursor ion peaks <NUM> and <NUM> of <FIG>. However, returning to <FIG>, XIC <NUM> and XIC <NUM> have very different relative intensities at times <NUM> and <NUM>. For example, at time <NUM>, both XIC <NUM> and XIC <NUM> have similar intensities. At time <NUM>, however, XIC <NUM> has a much larger intensity than XIC <NUM>.

The differences in the relative intensities of these groups of product ions for different isomers of the precursor ion show that the relative intensities of these groups of product ions can be used to identify linkages of isomers. For example, as described above, the intensities of the product ions of the transitions producing XIC <NUM> are known to be suppressed for a2,<NUM> sialic acid linkages and the product ions of the transitions producing XIC <NUM> are known to be enhanced for a2,<NUM> sialic acid linkages. At time <NUM>, the intensity of XIC <NUM> is much greater than the intensity of XIC <NUM>. As a result, the isomer of the precursor ion at time <NUM> has an a2,<NUM> sialic acid linkage <NUM>.

Conversely, as described above, the intensities of the product ions of the transitions producing XIC <NUM> are known to be enhanced for a2,<NUM> sialic acid linkages and the product ions of the transitions producing XIC <NUM> are known to be suppressed for α2,<NUM> sialic acid linkages. At time <NUM>, the intensity of XIC <NUM> is the same or slightly less than the intensity of XIC <NUM>. As a result, the isomer of the precursor ion at time <NUM> has an a2,<NUM> sialic acid linkage <NUM>.

Consequently, <FIG> shows how a comparison of the relative intensities of two groups of one or more MRM transitions monitored using ECD with an electron energy of <NUM> eV can be used to distinguish peaks of precursor ion isomers and, in turn, precursor ion peaks with different sialic acid linkages. Abundances or quantities of the precursor ions with different sialic acid linkages calculated from their peaks can then be used to diagnose different pathophysiological processes.

One of ordinary skill in the art understands that a comparison of relative intensities of two groups is equivalent to a calculation of a ratio of the intensities of the two groups. Similarly, one of ordinary skill in the art understands that a comparison of abundances or quantities of two precursor ions is also equivalent to calculating a ratio of the quantities of the two precursor ions.

<FIG> relate to a glycopeptide with a single sialic acid in a two-antenna typed N glycan and, therefore, a single sialic acid linkage. In various embodiments, isomers of glycopeptides with two or more sialic acids in two-four antenna type N glycans and sialic acid linkages are identified similarly. However, these isomers are identified based on a combination of two or more sialic acid linkages selected from the set of α2,<NUM> and α2,<NUM> sialic acid linkages.

<FIG> is an exemplary diagram <NUM> showing a glycan with two sialic acids attached to a peptide backbone forming a glycopeptide, in accordance with various embodiments. In <FIG>, glycan <NUM> is attached at one end to peptide backbone <NUM>. Glycan <NUM> includes two antennae <NUM> and <NUM>, two Gal sugars <NUM> and <NUM>, and two sialic acids Neu5Ac <NUM> and <NUM>. A glycopeptide with the configuration of glycan <NUM> is referred to as an A2G2Sa2 glycopeptide, referring to two antennae, two Gal sugars, and two Neu5Ac type sialic acids, respectively.

In various embodiments, to determine the linkage of Neu5Ac <NUM> to Gal sugar <NUM> and the linkage of Neu5Ac <NUM> to Gal sugar <NUM> of each isomer, the isomers of the glycopeptide of <FIG> are subjected to MS as they are eluting from the LC column. The m/z of the precursor ion of the glycopeptide is <NUM>, for example. From the MS, a precursor XIC of the glycopeptide is obtained.

<FIG> is an exemplary plot <NUM> of a precursor XIC from MS of the glycopeptide of <FIG>, in accordance with various embodiments. Precursor XIC <NUM> includes three precursor ion peaks <NUM>, <NUM>, and <NUM> representing at least three isomers of the precursor ion. Because the glycopeptide includes two sialic acids, peaks <NUM>, <NUM>, and <NUM> are known to represent three different combinations of two selections from the set of a2,<NUM> and α2,<NUM> sialic acid linkages. More specifically, the three peaks represent the combinations (a2,<NUM>, a2,<NUM>), (a2,<NUM>, a2,<NUM>), and (α2,<NUM>, α2,<NUM>). To identify each peak with its combination of sialic acid linkages, two groups of product ions for the glycan of the glycopeptide of <FIG> are selected and monitored using ECD with an electron energy of <NUM> eV.

<FIG> is an exemplary plot <NUM> of two XICs measured for two different groups of MRM transitions developed for the glycopeptide of <FIG> and monitored using ECD with an electron energy of <NUM> eV, in accordance with various embodiments. XIC <NUM> represents a sum of the intensities measured for two MRM transitions <NUM> to <NUM> and <NUM>/z over a series of LC separation times. XIC <NUM> represents a sum of the intensities measured for two MRM transitions <NUM> to <NUM> and <NUM>/z over a series of LC separation times. MRM transitions <NUM> to <NUM> and <NUM>/z show a similar behavior as the <NUM>st group for sialic acid linkage diagnostics, and MRM transitions <NUM> to <NUM> and <NUM>/z show a similar behavior as the <NUM>nd group for sialic acid linkage diagnostics, so two peak intensities in the same group can be summed to increase the signal-to-noise ratio.

Both XIC <NUM> and XIC <NUM> produce product ion peaks at times <NUM>, <NUM>, and <NUM> corresponding to precursor ion peaks <NUM>, <NUM>, and <NUM> of <FIG>. However, returning to <FIG>, XIC <NUM> and XIC <NUM> have very different relative intensities at times <NUM>, <NUM>, and <NUM>. The differences in the relative intensities of these groups of product ions for different isomers of the precursor ion show that the relative intensities of these groups of product ions can be used to identify each isomer of the glycopeptide. In various embodiments, these differences in the relative intensities of these groups are expressed as a ratio and can be plotted as a function of the separation time.

<FIG> is an exemplary plot <NUM> of the precursor XIC of <FIG> superimposed on the ratio of the intensities of the two XICs of <FIG>, in accordance with various embodiments. Ratio <NUM> is the running or moving average of the sum of intensities from the two MRM transitions <NUM> to <NUM> and <NUM>/z (enhanced by α2,<NUM> linkages) divided by the running or moving average of the sum of intensities from the two MRM transitions <NUM> to <NUM> and <NUM> (enhanced by a2,<NUM> linkages). A moving average is used, for example, to smooth the intensities. However, a moving average is not mandatory.

Precursor XIC <NUM> is superimposed on ratio <NUM> to show how ratio <NUM> is used to identify the linkages of each peak of precursor XIC <NUM>. For example, the apex of precursor ion peak XIC of precursor XIC <NUM> is used to calculate a separation time <NUM> of a first isomer of the glycopeptide. Ratio <NUM>, at separation time <NUM>, is compared to predetermined ratio ranges or thresholds <NUM>, <NUM>, and <NUM>. Each range or threshold corresponds to a combination of a selection from the set of a2,<NUM> and a2,<NUM> sialic acid linkages taken one or more times. Selecting from the set one or more times correspond to the number of one or more sialic acids known to be included in the glycopeptide. The number of sialic acids is known from the m/z of the glycopeptide, for example.

In this case, the glycopeptide includes two sialic acids, so the selections are taken twice, meaning that each combination is a group of two sialic acid linkages. Specifically, ranges or thresholds <NUM>, <NUM>, and <NUM> correspond to linkage combinations (α2,<NUM>, α2,<NUM>) <NUM>, (α2,<NUM>, α2,<NUM>) <NUM>, and (α2,<NUM>, α2,<NUM>) <NUM>, respectively.

At separation time <NUM>, ratio <NUM> is near <NUM> and in range <NUM> corresponding to combination (α2,<NUM>, a2,<NUM>) <NUM>. In other words, the comparison of ratio <NUM> at separation time <NUM> to predetermined ratio ranges <NUM>, <NUM>, and <NUM> produces a match with combination (a2,<NUM>, a2,<NUM>) <NUM>. As a result, the first isomer, which is represented by peak <NUM> of precursor XIC <NUM>, is found to include two a2,<NUM> sialic acid linkages.

Similarly, the second isomer represented by peak <NUM> of precursor XIC <NUM> and found at separation time <NUM> is identified and found to include one a2,<NUM> sialic acid linkage and one a2,<NUM> sialic acid linkage (combination <NUM>). The third isomer represented by peak <NUM> of precursor XIC <NUM> and found at separation time <NUM> is identified and found to include two a2,<NUM> sialic acid linkages (combination <NUM>).

In various embodiments, a quantity, a maximum intensity, or some other feature of the precursor ion peaks of at least two isomers of the glycopeptide are calculated and compared to provide, a diagnosis of a pathophysiological process. For example, the quantity of the first isomer represented by peak <NUM> is compared to the quantity of the second isomer represented by peak <NUM>. In other words, a ratio of the two quantities is calculated. Using this quantity ratio and the identified combination of sialic acid linkages included by each isomer a diagnosis of a pathophysiological process is provided. The diagnosis is provided, for example, by using a set of rules relating the quantity ratio and combination of sialic acid linkages to one or more pathophysiological processes.

In various embodiments, CID is used in addition to ECD to identify one or more linkages of a sialic acid to a sugar of an isomer other than Gal. For example, using CID, a group of one or more MRM transitions are monitored, producing an XIC for each transition of the group. The transitions of the group are known to fragment linkages of a sialic acid to a sugar other than Gal. These linkages are then identified from the XICs.

<FIG> is an exemplary plot <NUM> of a precursor XIC superimposed on the ratio of intensities of two XICs found using ECD that shows isomers found using both ECD and CID, in accordance with various embodiments. <FIG> shows eight isomers <NUM>-<NUM> identified for a glycopeptide using both ECD and CID. More specifically, eight different sialic acid to sugar linkages are identified for isomers <NUM>-<NUM>.

The glycan of the glycopeptide of <FIG> includes three antennae, three Gal sugars, and three sialic acids. A glycopeptide with the configuration of this glycan is referred to as an A3G3Sa3 glycopeptide.

The glycan of the glycopeptide of <FIG> is known, for example, to include linkages of sialic acid to Man in addition to linkages of sialic acid to Gal. As a result, CID is performed in addition to ECD. CID is used to identify linkages of sialic acid to Man.

ECD with an electron energy of <NUM> eV is used to produce ratio <NUM> as described above. Precursor XIC <NUM> is superimposed on ratio <NUM> and both are used, as described above, to identify sialic acid to Gal linkages. By identifying sialic acid to Man using CID and by identifying sialic acid to Gal linkages using ECD, all eight isomers <NUM>-<NUM> are identified. Note that more than one isomer is identified for two precursor ion peaks of precursor XIC <NUM>.

<FIG> is a schematic diagram <NUM> of a system for identifying one or more linkages of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, in accordance with various embodiments. The system of <FIG> includes separation device <NUM>, ion source <NUM>, tandem mass spectrometer <NUM>, and processor <NUM>.

Separation device <NUM> separates one or more isomers <NUM> of a glycopeptide from a sample. One or more isomers <NUM> are digested from a glycoprotein, for example. Separation device <NUM> can separate one or more isomers <NUM> using any separation technique including, but not limited to, LC, CE, or IMS.

Ion source <NUM> ionizes the one or more separated isomers, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide. Ion source <NUM> is shown as performing electrospray ionization (ESI) (e.g., nanospray) but can be any type of ion source. Ion source <NUM> is shown as part of tandem mass spectrometer <NUM> but can also be a separate device.

Tandem mass spectrometer <NUM> includes ECD device <NUM> and CID device <NUM>. For each separation time of a plurality separation times, tandem mass spectrometer <NUM> executes or monitors a first and a second group of MRM transitions using ECD device <NUM> with an electron energy of <NUM>-<NUM> eV. The first group of one or more MRM transitions is selected so that each transition includes the precursor ion and a product ion known to be enhanced or suppressed for a first linkage of a sialic acid of the glycopeptide to a glycan. The second group of one or more MRM transitions is selected so that each transition includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage of the sialic acid of the glycopeptide to the glycan. Precursor XIC <NUM> is produced for the precursor ion. An XIC is produced for each product ion of the first and second groups as shown in plot <NUM>.

Processor <NUM> is in communication with separation device <NUM>, ion source <NUM>, and tandem mass spectrometer <NUM>. Processor <NUM> performs a number of steps. Processor <NUM> calculates a separation time of an isomer of one or more separated isomers <NUM> from a peak of precursor XIC <NUM>. Processor <NUM> sums product ion intensities of the first group at the separation time producing a first sum and sums product ion intensities of the second group at the separation time producing a second sum using XICs of the first and second groups. Processor <NUM> calculates a ratio of the first sum to the second sum. Processor <NUM> compares the ratio at the separation time to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times (plot <NUM>). The one or more times correspond to the number of one or more sialic acids known to be included in the glycopeptide. Processor <NUM> identifies one or more linkages of the sialic acid to the glycan of the isomer from the combination found to match the ratio in the comparison. In other words, one or more linkages of the sialic acid to the glycan are identified for each peak of the precursor XIC as shown in plot <NUM>.

In various embodiments, the sialic acid includes Neu5Ac, the glycan includes Gal, the first linkage includes Neu5Ac-alpha (<NUM>,<NUM>) to Gal (α2,<NUM>) and the second linkage includes Neu5Ac-alpha (<NUM>,<NUM>) to Gal (α2,<NUM>).

In various embodiments, the sialic acid includes Neu5Gc and the glycan includes Man.

In various embodiments, tandem mass spectrometer <NUM> fragments the precursor ion of the first group of transitions and the second group of transitions using ECD device <NUM> with an electron energy of <NUM> eV.

In various embodiments, the one or more transitions of the first group can include a product ion of <NUM>/z, product ions of <NUM>, <NUM>, and <NUM>/z, product ions of <NUM>, <NUM> and <NUM>/z, or product ions of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>/z.

In various embodiments, the one or more transitions of the second group can include a product ion of <NUM>/z, product ions of <NUM>, <NUM>, and <NUM>/z, product ions of <NUM>, <NUM>, and <NUM>/z, or product ions of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>/z.

In various embodiments, in step B, processor <NUM> sums a moving average of product ion intensities of the first group at the separation time producing the first sum and sums a moving average of product ion intensities of the second group at the separation time producing the second sum using XICs of the first and second groups.

In various embodiments, processor <NUM> further calculates a separation time of a second isomer of the one or more separated isomers from a second peak of precursor XIC <NUM> and performs steps B-E for the second isomer to identify one or more linkages of the sialic acid to glycan of the second isomer.

In various embodiments, processor <NUM> calculates a first quantity of the isomer from the separation time of the first isomer and precursor XIC <NUM> and a second quantity of the second isomer from the separation time of the second isomer and precursor XIC <NUM>. Processor <NUM> further calculates a ratio of the first quantity to the second quantity. Processor <NUM> provides a diagnosis of a pathophysiological process based on the ratio, the identified one or more linkages of the sialic acid to the glycan of the isomer, and the identified one or more linkages of the sialic acid to the glycan of the second isomer.

In various embodiments, the pathophysiological process can include, but is not limited to, one or more of a viral infection, embryogenesis, inflammation, a cardiovascular disease, a cancer, or a neural development condition.

In various embodiments, tandem mass spectrometer <NUM> further, for each separation time of the plurality of separation times, using CID device <NUM>, executes on the ion beam a third group of one or more MRM transitions. An XIC is produced for each product ion of the third group. Processor <NUM> further identifies one or more linkages of the sialic to another glycan of the isomer from XICs of the third group.

In various embodiments, processor <NUM> is used to control or provide instructions to separation device <NUM>, ion source <NUM>, and tandem mass and to analyze data collected. Processor <NUM> controls or provides instructions by, for example, controlling one or more voltage, current, or pressure sources (not shown). Processor <NUM> can be a separate device as shown in <FIG> or can be a processor or controller of one or more devices of tandem mass spectrometer <NUM>, for example. Processor <NUM> can be, but is not limited to, a controller, a computer, a microprocessor, the computer system of <FIG>, or any device capable of sending and receiving control signals and data.

<FIG> is a flowchart showing a method <NUM> for identifying one or more linkages of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, in accordance with various embodiments.

In step <NUM> of method <NUM>, a separation time of an isomer of one or more isomers of a glycopeptide of a sample is calculated from a peak of a precursor XIC using a processor. The one or more isomers are separated from the sample using a separation device. The one or more separated isomers are ionized using an ion source, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide. For each separation time of a plurality separation times, using an ECD device with <NUM>-<NUM> eV electrons, a first and second group of one or more MRM transitions are monitored or executed on the ion beam. Each transition of the first group of one or more MRM transitions includes the precursor ion and a product ion known to be enhanced or suppressed for a first linkage of a sialic acid of the glycopeptide to a glycan of the glycopeptide. Each transition of the second group of one or more MRM transitions includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage for the sialic acid to the glycan. The precursor XIC is produced for the precursor ion and an XIC is produced for each product ion of the first and second groups.

In step <NUM>, product ion intensities of the first group are summed at the separation time producing a first sum and product ion intensities of the second group are summed at the separation time producing a second sum using XICs of the first and second groups using the processor.

In step <NUM>, a ratio of the first sum to the second sum is calculated using the processor.

In step <NUM>, the ratio at the separation time is compared to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times using the processor. The one or more times correspond to one or more sialic acids known to be included in the glycopeptide.

In step <NUM>, one or more linkages of the sialic acid to the glycan of the isomer are identified from a combination found to match the ratio in the comparison using the processor.

In various embodiments, computer program products include a tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for identifying one or more linkages of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions. This method is performed by a system that includes one or more distinct software modules.

<FIG> is a schematic diagram of a system <NUM> that includes one or more distinct software modules that perform a method for identifying one or more linkages of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, in accordance with various embodiments. System <NUM> includes an analysis module <NUM>.

Analysis module <NUM> calculates a separation time of an isomer of one or more isomers of a glycopeptide of a sample from a peak of a precursor XIC. The one or more isomers are separated from the sample using a separation device. The one or more separated isomers are ionized using an ion source, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide. For each separation time of a plurality separation times, using an ECD device with <NUM>-<NUM> eV electrons, a first and second group of one or more MRM transitions are monitored or executed on the ion beam. Each transition of the first group of one or more MRM transitions includes the precursor ion and a product ion known to be enhanced or suppressed for a first linkage of a sialic acid of the glycopeptide to a glycan of the glycopeptide. Each transition of the second group of one or more MRM transitions includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage for the sialic acid to the glycan. The precursor XIC is produced for the precursor ion and an XIC is produced for each product ion of the first and second groups.

Analysis module <NUM> sums product ion intensities of the first group at the separation time producing a first sum and sums product ion intensities of the second group at the separation time, producing a second sum using XICs of the first and second groups. Analysis module <NUM> calculates a ratio of the first sum to the second sum. Analysis module <NUM> compares the ratio at the separation time to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times. The one or more times correspond to one or more sialic acids known to be included in the glycopeptide. Finally, analysis module <NUM> identifies one or more linkages of the sialic acid to the glycan of the isomer from a combination found to match the ratio in the comparison.

Claim 1:
A system for identifying one or more linkages of a sialic acid (SA) to a glycan of an isomer of a glycopeptide using electron capture dissociation (ECD) and multiple reaction monitoring (MRM) precursor ion to product ion transitions, said system comprising:
a separation device configured to separate one or more isomers of a glycopeptide from a sample;
an ion source configured to ionize the one or more separated isomers, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide;
a tandem mass spectrometer that is configured to, for each separation time of a plurality separation times, using an ECD device with <NUM>-<NUM> eV electrons, execute on the ion beam a first group of one or more MRM transitions that each includes the precursor ion and a product ion known to be enhanced or suppressed for a first linkage of an SA of the glycopeptide to a glycan of the glycopeptide and a second group of one or more MRM transitions that each includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage for the SA to the glycan, producing an extracted ion chromatogram (XIC) for the precursor ion and an XIC for each product ion of the first and second groups; and
a processor in communication with the tandem mass spectrometer that is configured to:
a. calculate a separation time of an isomer of the one or more separated isomers from a peak of the TIC,
b. sum product ion intensities of the first group at the separation time producing a first sum and sum product ion intensities of the second group at the separation time producing a second sum using XICs of the first and second groups,
c. calculate a ratio of the first sum to the second sum,
d. compare the ratio at the separation time to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times, wherein one or more times correspond to one or more SAs known to be included in the glycopeptide, and
e. identify one or more linkages of the SA to the glycan of the isomer from a combination found to match the ratio in the comparison.