Methods of identifying the glycosidic linkage of sialic acid in glycopeptides

Abstract

The isolation time of the isomer in the one or more isomers of the sialic acid glycopeptide of the sample is calculated from the peak of the precursor XIC. Using the first and second sets of XICs, the product ion intensities of the first set at the isolation time are summed to produce a first sum and the product ion intensities of the second set at the isolation time are summed to produce a second sum. The ratio of the first sum to the second sum is calculated. The ratio at the isolation time is compared to a predetermined range of ratios, each corresponding to a combination of one or more selections from the set of first and second bonds. The one or more bonds of the sialic acid and glycan of the isomer are identified from the combination found in the comparison that matches the ratio.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 049,157, filed July 8, 2020, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0003] The teachings herein relate to operating an electron capture dissociation (ECD) device of a tandem mass spectrometer to identify one or more sialic acid bonds of a glycopeptide. More specifically, the teachings herein relate to systems and methods for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using ECD with an electron energy of 2-5 eV and multiple reaction monitoring (MRM) precursor ion to product ion transitions. The systems and methods disclosed herein can be performed in conjunction with a processor, controller, microcontroller, or computer system, such as the computer system of Figure 1 BACKGROUND

[0004] Sialic acid linkage context

[0005] Sialic acid complex carbohydrates are known to play a key role in a variety of pathophysiological processes, including viral infection, embryogenesis, inflammation, cardiovascular disease, cancer, and neurodevelopment. The most common mammalian sialic acid contains different bonds of N-acetylneuraminic acid (Neu5Ac). These bonds include Neu5Ac-alpha(2,6) galactose (Gal) (hereinafter “a2,6”) and Neu5Ac-alpha(2,3) Gal (hereinafter “a2,3”). Determining the relative abundance of a2,6 and a2,3 is important for diagnosing the pathophysiological processes just described.

[0006] The stereochemical structure identification of a2,6 and a2,3 in N-glycans or N-glycopeptides is typically reported by separation techniques such as capillary electrophoresis (CE) and ion mobility (IMS). Unfortunately, no methods have been reported for the liquid chromatography coupled mass spectrometry (LC-MS) analysis of non-derivatized glycopeptides by conventional proteomic shotgun sample preparation. The use of LC-MS and shotgun sample preparation (non-derivatized glycopeptides) reduces experimental complexity and improves overall sample throughput. In contrast, biochemical identification employs bond-specific sialic acid derivatization of glycans prior to MS. In other words, these techniques typically require the chemical conversion of glycans to derivatives prior to mass spectrometry analysis (MS).

[0007] ​For example, Reiding et al., “High-throughput analysis of protein N-glycosylation by MALDI-TOF-MS employing key-specific sialylation” (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 uses a carboxylic acid activator in conjunction with ethanol to achieve near-complete ethyl esterification of a2,6 sialic acids and lactonization of a2,3 variants. In other words, the Reiding paper describes an ethyl esterification chemical transformation of glycans prior to MS.

[0008] Similarly, Zhao et al., “Collisional activation dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides” (hereinafter the “Zhao paper”) describes a method for confirming the sequence, branching, and linkage assignment of glycans. The Zhao paper does not involve determining a2,6 and a2,3 linkages. However, the Zhao paper does describe subjecting glycans to permethylation to increase their sensitivity to collisional activation dissociation (CAD) and electron capture dissociation (ECD). In other words, the Zhao paper describes a permethylation chemical transformation of glycans prior to MS. For these glycans, CAD and “hot” ECD provide complementary structural information.

[0009] Thus, there is a need for systems and methods for determining the abundance of the sialic acid linkages a2,6 and a2,3 of glycopeptides using LC-MS or LC-MS / MS of non-derivatized glycopeptides prepared by conventional proteomic shotgun sample preparation.

[0010] Mass spectrometry analysis technique context

[0011] Mass spectrometers are often coupled with a chromatography or other separation system, such as ion mobility, in order to identify and characterize known compounds of interest from a sample. In such coupled systems, the elution solvent is ionized and a series of mass spectra are obtained from the elution solvent at specific time intervals known as retention times. These retention times range from, for example, 1 second to 100 minutes or more. The series of ion intensities measured at the retention times form a chromatogram, or extracted ion chromatogram (XIC).

[0012] The peaks found in the XIC are used to identify or characterize known peptides or compounds in the sample. More specifically, the retention times of the peaks and / or the areas of the peaks are used to identify or characterize (quantify) the known peptides or compounds in the sample.

[0013] In traditional separated coupled mass spectrometry systems, fragment or product ions of known compounds are selected for analysis. A tandem mass spectrometry or mass spectrometry / mass spectrometry (MS / MS) scan is then performed for each interval of separation against a mass range that includes the product ions. The intensities of the product ions found in each MS / MS scan are collected over time and analyzed as, for example, a spectral library or an XIC.

[0014] Generally, 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.

[0015] Tandem mass spectrometry can provide both qualitative and quantitative information. Product ion spectra can be used to identify molecules of interest. The intensities of one or more product ions can be used to quantify the amount of a compound present in a sample.

[0016] A 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).

[0017] In a targeted acquisition method, one or more transitions of precursor ions to product ions are predefined for a compound of interest. When the sample is introduced into the tandem mass spectrometer, the one or more transitions are interrogated or monitored during each of a number of time periods or cycles. In other words, the mass spectrometer selects and fragments the precursor ion of each transition and performs targeted mass analysis only of the product ions of that transition. As a result, an intensity (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).

[0018] Multiple reaction monitoring (MRM) on a triple quadrupole based instrument is the standard mass spectrometry technique of choice for targeted MS quantification in all fields of application as it enables 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 has enabled new quantification strategies with similar performance characteristics to become possible. In this strategy, referred to as the MRM-HR workflow or parallel reaction monitoring, PRM, circular MS / MS spectra are collected at high resolution and short accumulation times, then fragment ions are extracted post-acquisition to generate MRM-like peaks for integration and quantification. With instruments like the Orbitrap® system, this targeted technique is very sensitive and fast enough to enable similar quantification performance to higher end triple quadrupole instruments and enables the measurement of complete fragmentation data at high resolution and high mass accuracy. With instruments such as the Orbitrap® system, this targeted technique is very sensitive and fast enough to enable similar quantification performance to higher end triple quadrupole instruments and enables the measurement of complete fragmentation data at high resolution and high mass accuracy.

[0019] In IDA methods, a user can specify criteria for performing non-targeted mass analysis of product ions while a sample is introduced into a tandem mass spectrometer. For example, in IDA methods, a precursor ion or mass spectrometry (MS) survey scan is performed to generate a list of precursor ion peaks. A user can select criteria to filter the list of peaks to a subset of precursor ions on the list of peaks. MS / MS is then performed for each precursor ion in the subset of precursor ions. A product ion spectrum is produced for each precursor ion. When a sample is introduced into a tandem mass spectrometer, MS / MS is repeatedly performed for the precursor ions of the subset of precursor ions.

[0020] However, in proteomics and many other sample types, the complexity and dynamic range of compounds is very large. This presents a challenge to traditional targeted methods and IDA methods, which require very high speed MS / MS acquisition to deeply interrogate a sample in order to identify and quantify a broad range of analytes.

[0021] Accordingly, a third broad class of tandem mass spectrometry methods, DIA methods, has been developed. These DIA methods have been used to improve the reproducibility and comprehensiveness of data collected from complex samples. DIA methods can also be referred to as non-specific fragmentation methods. In traditional DIA methods, the action of the tandem mass spectrometer does not change between MS / MS scans as a function of data obtained in previous precursor ion scans or product ion scans. Rather, 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 product ions of all precursor ions in the precursor ion mass selection window are mass analyzed.

[0022] The precursor ion mass selection window used to scan a mass range can be very narrow, such that there is little likelihood of multiple precursors existing within the window. For example, this type of DIA method is referred to as MS / MS ALL . In MS / MS ALL methods, a precursor ion mass selection window of about 1 amu is scanned or stepped across the entire mass range. A product ion spectrum is produced for each 1 amu precursor mass window. The time required to analyze or scan the entire mass range once is referred to as one scan cycle. However, scanning a narrow precursor ion mass selection window across a wide precursor ion mass range during each cycle is not practical for some instruments and experiments.

[0023] Accordingly, larger precursor ion mass selection windows or selection windows with larger widths are stepped across the entire precursor mass range. This type of DIA method is referred to as, for example, SWATH acquisition. In SWATH acquisition, the precursor ion mass selection windows stepped across the precursor mass range in each cycle can have a width of 5-25 amu or even larger. With MS / MSALL As with the method, all precursor ions in each precursor ion mass selection window are fragmented and all product ions for all precursor ions in each mass selection window are mass analyzed.

[0024] Fragmentation technique context

[0025] Electron-based dissociation (ExD), ultraviolet photodissociation (UVPD), infrared photodissociation (IRMPD), and collision-induced dissociation (CID) or CAD are commonly 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 traditional dissociation technique in tandem mass spectrometers.

[0026] As described above, in top-down and middle-down proteomics, intact or digested proteins are ionized and subjected to tandem mass spectrometry. For example, ECD is a dissociation technique that preferentially dissociates peptide and protein backbone. Thus, this technique is an ideal tool for analyzing peptide or protein sequences using top-down and middle-down proteomics methods. SUMMARY

[0027] According to various embodiments, a system, method, and computer program product are disclosed for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions. The system includes a separation device, an ion source, a tandem mass spectrometer, and a processor.

[0028] The separation device separates one or more isomers of a glycopeptide from a sample. The ion source ionizes the separated one or more isomers, thereby producing an ion beam comprising isomer ions of a precursor ion of the glycopeptide.

[0029] The tandem mass spectrometer includes an ECD device. For each separation time of a plurality of separation times, the tandem mass spectrometer performs or monitors a first set of MRM transitions and a second set of MRM transitions using the ECD device with an electron energy of 2-5 eV. The first set of one or more MRM transitions is selected such that each transition includes a precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of the glycopeptide. The second set of one or more MRM transitions is selected such that each transition includes the precursor ion and a product ion known to be enhanced or inhibited for a second bond of the sialic acid to the glycan of the glycopeptide. In various embodiments, MRM-HR can be used to monitor all product ions simultaneously. An XIC is generated for the precursor ion. An XIC is generated for each product ion of the first set and the second set.

[0030] The processor performs a plurality of steps. The processor calculates a separation time of an isomer of the one or more isomers separated from the peak of the TIC. The processor sums the product ion intensities of the first set at the separation time to produce a first sum and sums the product ion intensities of the second set at the separation time to produce a second sum using the XICs of the first set and the second set. The processor calculates a ratio of the first sum to the second sum. The processor compares the ratio at the separation time to a predetermined ratio range, each predetermined ratio range corresponding to a combination of one or more selections made from a set of the first bond and the second bond. The one or more corresponds to a number of sialic acids known to be included in the glycopeptide. Finally, the processor identifies the one or more bonds of the isomer of sialic acid to the glycan from the combination found to match the ratio in the comparison.

[0031] These and other features of the Applicant's teachings are set forth with particularity below. BRIEF DESCRIPTION OF DRAWINGS

[0032] Those skilled in the art will appreciate that the figures described below are for purposes of illustration only. The figures are not intended to limit the scope of the present teachings in any way.

[0033] Figure 1 is a block diagram illustrating a computer system on which embodiments of the present teachings can be implemented.

[0034] Figure 2 is an example diagram showing a structural formula and Oxford system notation for a glycan including two sialic acids, each having a different bond of sialic acid to sugar, in accordance with various embodiments.

[0035] Figure 3 is an example diagram showing a glycan having one sialic acid attached to a peptide backbone to form a glycopeptide, in accordance with various embodiments.

[0036] Figure 4 is an example plot of MS of a glycopeptide from Figure 3 , in accordance with various embodiments.

[0037] Figure 5 is an example plot of a series of two XICs measured for different two sets of MRM transitions developed for a glycopeptide that is Figure 3 , in accordance with various embodiments, monitored using ECD with electron energy of 3 eV.

[0038] Figure 6 is an example diagram showing a glycan having two sialic acids attached to a peptide backbone to form a glycopeptide, in accordance with various embodiments.

[0039] Figure 7 is an example plot of MS of a glycopeptide from Figure 6Example plot of precursor XIC of MS of glycopeptide.

[0040] Figure 8 Example plot of precursor XIC of MS of glycopeptide. Figure 6 Example plot of two XICs developed for glycopeptide of

[0041] Figure 9 Example plot of precursor XIC of MS of glycopeptide. Figure 7 Example plot of two XICs developed for glycopeptide of Figure 8

[0042] Figure 10 Example plot of precursor XIC of MS of glycopeptide.

[0043] Figure 11 Schematic of a system for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, according to various embodiments.

[0044] Figure 12 Flowchart showing a method for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, according to various embodiments.

[0045] Figure 13 Schematic of a system comprising one or more different software modules that perform a method for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions, according to various embodiments.

[0046] Before one or more embodiments of the present teachings are described in detail, it is to be understood that the application of the present teachings is not limited to the details of construction, the arrangements of components, and the arrangements of steps set forth in the following detailed description or illustrated in the drawings. Further, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. DETAILED DESCRIPTION

[0047] Computer implemented system

[0048] Figure 1 ​is a block diagram that illustrates a computer system 100 upon which an embodiment making use of the present teachings can be implemented. The computer system 100 includes a bus 102 or other communication mechanism for communicating information, and a processor 104 coupled with bus 102 for processing information. The computer system 100 also includes a memory 106, which can be random access memory (RAM) or other dynamic storage device, coupled to bus 102 for storing information and instructions to be executed by processor 104. Memory 106 also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 104. The computer system 100 further includes a read only memory (ROM) 108 or other static storage device coupled to bus 102 for storing static information and instructions for processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to bus 102 for storing information and instructions.

[0049] The computer system 100 can be coupled via bus 102 to a display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to bus 102 for communicating information and command selections to processor 104. Another type of user input device is cursor control 116, such as a mouse, a trackball, or cursor direction keys for communicating direction information and command selections to processor 104 and for

[0050] The computer system 100 can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system 100 in response to processor 104 executing one or more sequences of one or more instructions contained in memory 106. Such instructions can be read into memory 106 from another computer-readable medium, such as storage device 110. Execution of the sequences of instructions contained in memory 106 causes processor 104 to perform the process described herein. Alternatively, hard-wired circuitry can be used in place of software instructions, 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.

[0051] In various embodiments, computer system 100 can connect to one or more other computer systems, such as computer system 100, across a network to form a networked system. The network can include a private network or a public network, such as the Internet. In a networked system, one or more computer systems can store data and provide data to other computer systems. In a cloud-computing scenario, the one or more computer systems that store and provide data can be referred to as a server or a cloud. For example, the one or more computer systems can include one or more web servers. For example, other computer systems that send data to and receive data from the server or cloud can be referred to as clients or cloud devices.

[0052] The term“computer-readable medium” as used herein refers to any medium that participates in providing instructions to processor 104 for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 110. Volatile media include dynamic memory, such as memory 106. Transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102.

[0053] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, a hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disk (DVD), a Blu-ray disk, 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.

[0054] Various forms of computer-readable media can be involved in carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions can initially be carried on a magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 100 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 102 can receive the data carried in the infra-red signal and place the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. The instructions received by memory 106 can optionally be stored on storage device 110 either before or after execution by processor 104.

[0055] According to various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer readable medium. The computer readable medium can be a device that stores digital information. For example, the computer readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer readable medium is accessed by a processor suitable for executing the instructions configured to be executed.

[0056] The following description of various implementations of the present teachings has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the present teachings to the precise form disclosed. Many modifications and variations are possible in light of the above teachings, or can be acquired from practice of the present teachings. Further, the described implementations include software, but the present teachings can be implemented as a combination of hardware and software, or entirely in hardware. The present teachings can be realized in both object-oriented and non-object-oriented programming systems.

[0057] Identifying isomers with different sialic acid linkages

[0058] As described above, determining the relative abundance of a2, 6 and a2, 3 is important in diagnosing pathophysiological processes including, but not limited to, viral infection, embryogenesis, inflammation, cardiovascular disease, cancer, and neurodevelopment. The stereochemical structure identification of a2, 6 and a2, 3 N-glycans or N-glycopeptides is typically reported by separation techniques such as CE and IMS. Unfortunately, no methods have been reported using LC-MS or LC-MS / MS analysis of non-derivatized glycopeptides using conventional proteomic shotgun sample preparation. Using LC-MS and shotgun sample preparation (non-derivatized glycopeptides) reduces experimental complexity and improves overall sample throughput.

[0059] Accordingly, there is a need for systems and methods for determining the abundance of the sialic acid bond a2, 6 and a2, 3 of glycopeptides using LC-MS or LC-MS / MS of non-derivatized glycopeptides using conventional proteomic shotgun sample preparation.

[0060] In various embodiments, isomers of glycopeptides are identified using LC-MS / MS, the isomers having different combinations of the sialic acid bond a2, 6 and a2, 3 of the glycopeptides. The isomers are separated using LC. When the isomers elute, a set of MRM transitions targeting the a2, 6 and a2, 3 bonds are monitored using ECD at an electronic energy between 2-5 eV. The intensity ratio measured from the different two sets of transitions is used to identify the sialic bond of the isomers as a2, 6 or a2, 3. The relative abundance or amount of the isomers having different sialic bonds is then used to diagnose pathophysiological processes.

[0061] For example, a glycopeptide 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 variety of specific biological functions. The peptide backbone is an amino acid sequence. If a glycan is attached to an amino acid asparagine (N) of the peptide backbone, the peptide is referred to as an N-glycopeptide.

[0062] Figure 2 is an example diagram 200 showing a structural formula and Oxford system notation of a glycan that includes two sialic acids, each having a different sialic acid to sugar linkage. In Figure 2 the structural formula 210 shows a sialic acid Neu5Ac 211 attached at the end of one of the antennae of the glycan and another sialic acid Neu5Ac 212 attached at the end of the other of the antennae of the glycan. Neu5Ac 211 is attached to a Gal of the glycan by an a2,6 linkage 213. Neu5Ac 212 is attached to a Gal of the glycan by an a2,3 linkage 214.

[0063] Equivalently, the Oxford system notation 220 shows Neu5Ac 221 attached at the end of one of the antennae of the glycan and Neu5Ac 222 attached at the end of the other of the antennae of the glycan. Neu5Ac 221 is attached to a Gal 225 of the glycan by an a2,6 linkage 223. Neu5Ac 222 is attached to a Gal 226 of the glycan by an a2,3 linkage 224. In the Oxford system notation 220, the a2,6 linkage 223 and the a2,3 linkage 224 are curved in different directions relative to their antennae to indicate that they are different linkages. For example, at the end of the glycan opposite Neu5Ac 221 and Neu5Ac 222, the glycan is connected or attached to a backbone (not shown) of a peptide.

[0064] Other components of the glycan include mannose (Man) 227 and N-acetylglucosamine (GlcNAc) 228. Gal 225, Gal 226, and Man 227 are types of hexoses. GlcNAc 228 and N-acetylgalactosamine (GalNAc) are types of hexosamines (hexNAc). In Figure 2 in which the sialic acids are shown as Neu5Ac 221 and Neu5Ac 222. In various alternative embodiments, the sialic acids can be, but are not limited to, N-glycolylneuraminic acid (Neu5Gc or Sg). For example, humans do not have Sg.

[0065] In various embodiments, glycopeptides, such as N-glycopeptides, are found in digested proteins and separated by LC. For example, LC separation methods of glycopeptides include, but are not limited to, reversed-phase LC and non-reversed-phase hydrophilic interaction liquid chromatography (HILIC).

[0066] Figure 3Figure 300 illustrates examples of various embodiments where a glycan having a sialic acid atom is attached to the peptide backbone to form a glycopeptide. Figure 3 In this structure, glycan 310 is attached to the peptide backbone 320 at one end. As described above, the peptide backbone 320 consists of a series of one or more amino acids. Glycan 310 includes two tendon-like structures 311 and 312, two gal sugars 313 and 314, and a sialic acid Neu5Ac 315. Glycopeptides with the structure of glycan 310 are sometimes referred to as A2G2Sal glycopeptides, referring to the two tendon-like structures, two gal sugars, and one sialic acid.

[0067] Glycan 310 has only one sialic acid, Neu5Ac 315, and therefore represents the simplest case. Figure 3 In the diagram, the bonds of Neu5Ac 315 with Gal sugars 313 and 314 are not shown to be bent to the left or right. Therefore, it does not describe either of the two possible bond types, α2,6 or α2,3.

[0068] In various embodiments, in order to determine the bonds between Neu5Ac 315 and Gal sugars 313 and 314, Figure 3 The glycopeptide undergoes MS during elution from the LC column. For example, the m / z of the glycopeptide is 914.4. The precursor XIC of the glycopeptide is obtained from MS.

[0069] Figure 4 Based on various embodiments from Figure 3 Example graph 400 shows the MS precursor XIC of the glycopeptide. Precursor XIC401 includes... Figure 3 The two precursor ion peaks of the glycopeptide are 410 and 420. Figure 4 Peaks 410 and 420 in the spectrum represent isomers of the glycopeptide. In other words, they share the same m / z of 914.4, but their structures differ. Specifically, peaks 410 and 420 represent... Figure 3 The Neu5Ac 315 in the sugar differs from the 313 and 314 bonds in Gal sugar. In other words, back to... Figure 4 , Figure 4 One of the peaks at 410 and 420 represents an α2,6 bond, and the other peak represents an α2,3 bond.

[0070] As described above with reference to the Reiding paper and the Zhao paper, it is generally believed that the use of glycan bond-specific sialic acid derivatization prior to MS is not possible to distinguish between a2, 6 and a2, 3 linkages. More broadly, it is well known that ExD and CID are complementary for glycopeptide analysis. However, it is generally believed that ExD or ECD specifically acts on the peptide backbone in a glycopeptide, while CID acts on the glycan in a glycopeptide, although ECD has been shown to also act on free glycans. In general, it is well known that "low" ECD (0-2 eV) and ETD do not provide good dissociation efficiency, while "hot" ECD (5-10 eV) is better for more complex glycans. For example, the Zhao paper describes that hot ECD on a doubly charged disialylated glycan is not useful. In contrast, the Zhao paper reports that hot ECD provides more structural information than CID for larger, complex glycans.

[0071] In various embodiments, experiments have shown that ECD with electron energy between 2 and 5 eV, preferably 3 eV, produces detectable glycan fragments in a glycopeptide. These fragments are then used to distinguish between a2, 6 and a2, 3 sialic acid linkages of a glycan.

[0072] Returning to Figure 3 , it is shown that two groups of fragments or product ions 331 and 332 of the glycan of the glycopeptide of Figure 3 When this glycopeptide (914.4 m / z) is fragmented with ECD with electron energy of 3 eV, it is found that the product ions of group 331 are enhanced for a2, 6 sialic acid linkages, and it is found that the product ions of group 332 are suppressed for a2, 6 sialic acid linkages. Conversely, under the same conditions, it is found that the product ions of group 331 are suppressed for a2, 3 sialic acid linkages, and it is found that the product ions of group 332 are enhanced for a2, 3 sialic acid linkages. In other words, using ECD with electron energy of 3 eV, a2, 6 and a2, 3 sialic acid linkages can be identified by comparing the intensities of the different groups of product ions.

[0073] Figure 5 is an example series 500 of exemplary plot lines of two XICs measured according to various embodiments for a glycopeptide of Figure 3 Formula (I) using ECD with electron energy of 3 eV. The XIC 501 in plot line 531 represents the sum of the measured intensities of the three MRM transitions 914.4 to 676.5, 677.5, and 678.5 m / z measured at a series of LC separation times. The XIC 502 in plot line 532 represents the sum of the measured intensities of the three MRM transitions 914.4 to 292.1, 293.1, and 294.1 m / z measured at a series of LC separation times.

[0074] The XIC 501 and the XIC 502 are measured at a series of LC separation times corresponding to the elution of the glycopeptide of Figure 4product ion peaks at times 510 and 520. However, returning to Figure 5 XIC 501 and XIC 502 have very different relative intensities at times 510 and 520. For example, at time 510, both XIC 501 and XIC 502 have similar intensities. However, at time 520, the intensity of XIC 502 is much greater than that of XIC 501.

[0075] The difference in the relative intensities of these product ion groups for different isomers of the precursor ion indicates that the relative intensities of these product ion groups can be used to identify the linkage of the isomer. For example, as noted above, the intensity of the product ion that produces XIC 501 is known to be suppressed for an a2,3 sialic acid linkage, and the intensity of the product ion that produces XIC 502 is known to be enhanced for an a2,3 sialic acid linkage. At time 520, the intensity of XIC 502 is much greater than that of XIC 501. Thus, the isomer of the precursor ion at time 520 has an a2,3 sialic acid linkage 521.

[0076] In contrast, as noted above, the intensity of the product ion that produces XIC 501 is known to be enhanced for an a2,6 sialic acid linkage, and the intensity of the product ion that produces XIC 502 is known to be suppressed for an a2,6 sialic acid linkage. At time 510, the intensity of XIC 502 is the same as or slightly less than that of XIC 501. Thus, the isomer of the precursor ion at time 510 has an a2,6 sialic acid linkage 511.

[0077] Thus, Figure 5 A comparison of the relative intensities of the two sets of one or more MRM transitions monitored using an ECD with an electron energy of 3 eV is shown to be useful to distinguish the peaks of the isomers of the precursor ion, and in turn, to distinguish the peaks of the precursor ions with different sialic acid linkages. The abundances or amounts of the precursor ions with different sialic acid linkages calculated from their peaks can then be used to diagnose different pathophysiological processes.

[0078] It is understood by one of ordinary skill in the art that comparing the relative intensities of the two sets is equivalent to calculating the intensity ratio of the two sets. Similarly, it is understood by one of ordinary skill in the art that comparing the abundances or amounts of the two precursor ions is also equivalent to calculating the amount ratio of the two precursor ions.

[0079] Figures 3-5glycopeptides having a single sialic acid and thus a single sialic acid linkage in a biantennary N-glycan. In various embodiments, isomers of glycopeptides having two or more sialic acids and sialic acid linkages in a di- to tetra-antennary N-glycan are similarly identified. However, these isomers are identified based on a combination of two or more sialic acid linkages selected from the set of a2,6 and a2,3 sialic acid linkages.

[0080] Figure 6 is an example plot 600 showing a glycan having two sialic acids attached to a peptide backbone to form a glycopeptide, according to various embodiments. In Figure 6 glycan 610 is attached to peptide backbone 620 at one end. Glycan 610 includes two antennae 611 and 612, two Gal sugars 613 and 614, and two sialic acids Neu5Ac 615 and 616. A glycopeptide having the configuration of glycan 610 is referred to as an A2G2Sa2 glycopeptide, referring to two antennae, two Gal sugars, and two Neu5Ac-type sialic acids, respectively.

[0081] In various embodiments, to determine the linkage of Neu5Ac 615 to Gal sugar 613 and the linkage of Neu5Ac 616 to Gal sugar 614 for each isomer, Figure 6 isomers of the glycopeptide of

[0082] Figure 7 is an example plot line 700 of a precursor XIC of MS of the glycopeptide of Figure 6 Precursor XIC 701 includes three precursor ion peaks 710, 720, and 730 representing at least three isomers of the precursor ion. Because the glycopeptide includes two sialic acids, peaks 710, 720, and 730 are known to represent three different combinations of two selections from the set of a2,6 and a2,3 sialic acid linkages. More specifically, the three peaks represent the combinations (a2,6, a2,6), (a2,6, a2,3), and (a2,3, a2,3). To identify each peak and its combination of sialic acid linkages, two sets of product ions of the glycan of the glycopeptide of Figure 6

[0083] Figure 8 is an example plot line 800 of a precursor XIC of MS of the glycopeptide of Figure 6 ​Figure 8 is an example plot 800 of two XICs measured for different two groups of MRM transitions for glycopeptide development and monitored using ECD with an electron energy of 3 eV. XIC 801 represents the sum of intensities measured for two MRM transitions 987.2 to 675 and 838 m / z at a series of LC separation times. XIC 802 represents the sum of intensities measured for two MRM transitions 987.2 to 274 and 292 m / z at a series of LC separation times. MRM transitions 987.2 to 675 and 838 m / z exhibit similar behavior to Group 1 for sialic acid linkage diagnostics, while MRM transitions 987.2 to 274 and 292 m / z exhibit similar behavior to Group 2 for sialic acid linkage diagnostics, so the intensities of the two peaks in the same group can be summed to improve signal-to-noise.

[0084] XIC 801 and XIC 802 both produce product ion peaks at times 810, 820, and 830 corresponding to precursor ion peaks 710, 720, and 730. Figure 7 However, returning to Figure 8 , XIC 801 and XIC 802 have very different relative intensities at times 810, 820, and 830. The difference in relative intensities of these product ion groups for different isomers of the precursor ion indicates that the relative intensities of these product ion groups can be used to identify each isomer of the glycopeptide. In various embodiments, these differences in relative intensities of these groups are expressed as ratios, and can be plotted as a function of separation time.

[0085] Figure 9 Figure 9 is an example plot 900 of a precursor XIC for Figure 7 superimposed on the intensity ratio of two XICs for Figure 8 . Ratio 901 is the running average or moving average of the sum of intensities from two MRM transitions 987.2 to 675 and 838 m / z (enhanced by a2,6 linkages) divided by the running average or moving average of the sum of intensities from two MRM transitions 987.2 to 274 and 292 (enhanced by a2,3 linkages). For example, moving averages are used to smooth the intensities. However, moving averages are not mandatory.

[0086] The precursor XIC 701 is overlaid on the ratio 901 to show how the ratio 901 is used to identify the linkage for each peak of the precursor XIC 701. For example, the apex of the precursor ion peak XIC of the precursor XIC 701 is used to calculate a retention time 910 of the first isomer of the glycopeptide. At the retention time 910, the ratio 901 is compared to predetermined ratio ranges or thresholds 941, 942, and 943. Each range or threshold corresponds to a combination of one or more selections from a set of a2,6 and a2,3 sialic acid linkages. The one or more selections from the set corresponds to a number of sialic acids known to be included in the glycopeptide. For example, the number of sialic acids is known from the m / z of the glycopeptide.

[0087] In this case, the glycopeptide includes two sialic acids, so two selections are made, meaning that each combination is a group of two sialic acid linkages. Specifically, the ranges or thresholds 941, 942, and 943 correspond to the linkage combinations (a2,6, a2,6) 951, (a2,6, a2,3) 952, and (a2,3, a2,3) 953, respectively.

[0088] At the retention time 910, the ratio 901 is close to 2.0 and in the range 941 corresponding to the combination (a2,6, a2,6) 951. In other words, the comparison of the ratio 901 at the retention time 910 to the predetermined ratio ranges 941, 942, and 943 yields a match to the combination (a2,6, a2,6) 951. Thus, the first isomer (represented by the peak 710 of the precursor XIC 701) is found to include two a2,6 sialic acid linkages.

[0089] Similarly, the second isomer (represented by the peak 720 of the precursor XIC 701) found at the retention time 920 is identified and found to include one a2,6 sialic acid linkage and one a2,3 sialic acid linkage (combination 952). The third isomer (represented by the peak 730 of the precursor XIC 701) found at the retention time 930 is identified and found to include two a2,3 sialic acid linkages (combination 953).

[0090] In various embodiments, the amounts, maximum intensities, or some other characteristic 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 amount of the first isomer represented by the peak 710 is compared to the amount of the second isomer represented by the peak 720. In other words, a ratio of these two amounts is calculated. Using this amount ratio and the identified combination of sialic acid linkages included by each isomer, a diagnosis of a pathophysiological process is provided. For example, the diagnosis is provided by using a set of rules that associate the amount ratio and the combination of sialic acid linkages to one or more pathophysiological processes.

[0091] In various embodiments, CID is used in addition to ECD to identify one or more bonds of the sialic acid to sugars other than Gal of the isomer. For example, using CID, a set of one or more MRM transitions is monitored, resulting in an XIC for each transition of the set. The transitions of the set are known to cleave a bond of the sialic acid to a sugar other than Gal. These bonds are then identified from the XICs.

[0092] Figure 10 is an example plot 1000 of superimposing a precursor XIC on a ratio of intensities of two XICs discovered using ECD according to various embodiments, showing isomers discovered using both ECD and CID. Figure 10 shows eight isomers 1010-1080 of a glycopeptide identified using both ECD and CID. More specifically, eight different bonds of sialic acid to sugar are identified for isomers 1010-1080.

[0093] Figure 10 The glycan of the glycopeptide of

[0094] The glycan of the glycopeptide of Figure 10 The glycan of the glycopeptide of includes a bond of sialic acid to Man in addition to bonds of sialic acid to Gal. Accordingly, CID is performed in addition to ECD. CID is used to identify the bond of sialic acid to Man.

[0095] ECD with an electron energy of 3 eV is used to produce the ratio 1001 as described above. The precursor XIC 1002 is superimposed on the ratio 1001, and both are used to identify the bonds of sialic acid to Gal as described above. By using CID to identify the bond of sialic acid to Man and by using ECD to identify the bonds of sialic acid to Gal, all eight isomers 1010-1080 are identified. Note that more than one isomer is identified for both precursor ion peaks of the precursor XIC 1002.

[0096] System for identifying sialic acid linkages

[0097] Figure 11 is a schematic diagram 1100 of a system for identifying one or more bonds of sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor ion to product ion transitions according to various embodiments. Figure 11 The system of includes a separation device 1110, an ion source 1120, a tandem mass spectrometer 1130, and a processor 1140.

[0098] The separation device 1110 separates one or more isomers 1101 of glycopeptides from a sample. For example, the one or more isomers 1101 are digested from a glycoprotein. The separation device 1110 can use any separation technique to separate the one or more isomers 1101, including but not limited to LC, CE, or IMS.

[0099] The ion source 1120 ionizes the separated one or more isomers, producing an ion beam comprising isomeric ions of precursor ions of glycopeptides. The ion source 1120 is shown as performing electrospray ionization (ESI) (e.g., nanospray) but can be any type of ion source. The ion source 1120 is shown as part of the tandem mass spectrometer 1130 but can also be a separate device.

[0100] The tandem mass spectrometer 1130 includes an ECD device 1131 and a CID device 1132. For each separation time of the plurality of separation times, the tandem mass spectrometer 1130 performs or monitors a first set of MRM transitions and a second set of MRM transitions using the ECD device 1131 with an electron energy of 2-5 eV. The first set of one or more MRM transitions is selected such that each transition includes a precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of the glycopeptide. The second set of one or more MRM transitions is selected such that each transition includes the precursor ion and a product ion known to be enhanced or inhibited for a second bond of the sialic acid to the glycan of the glycopeptide. A precursor XIC 1135 is generated for the precursor ion. As shown by plot 1136, an XIC is generated for each product ion of the first set and the second set.

[0101] The processor 1140 communicates with the separation device 1110, the ion source 1120, and the tandem mass spectrometer 1130. The processor 1140 performs a plurality of steps. A, the processor 1140 calculates a separation time of an isomer of the separated one or more isomers 1101 from a peak of the precursor XIC 1135. B, the processor 1140 sums product ion intensities of the first set at the separation time using the XICs of the first set and the second set, generating a first sum, and sums product ion intensities of the second set at the separation time, generating a second sum. C, the processor 1140 calculates a ratio of the first sum to the second sum. D, the processor 1140 compares the ratio at the separation time to a predetermined ratio range, each predetermined ratio range corresponding to a combination of one or more selections from a set of the first bond and the second bond (plot 1141). The one or more corresponds to a number of sialic acids known to be included in the glycopeptide. E, the processor 1140 identifies one or more bonds of sialic acids to glycans of the isomer from the combination found to match the ratio in the comparison. In other words, for each peak of the precursor XIC as shown by plot 1142, one or more bonds of sialic acids to glycans are identified.

[0102] In various embodiments, the sialic acid comprises Neu5Ac, the glycan comprises Gal, the first linkage comprises Neu5Ac-alpha (2,6) to Gal (a2,6), and the second linkage comprises Neu5Ac-alpha (2,3) to Gal (a2,3).

[0103] In various embodiments, the sialic acid comprises Neu5Gc and the glycan comprises Man.

[0104] In various embodiments, tandem mass spectrometer 1130 fragments the precursor ions of the first set of transitions and the second set of transitions using ECD device 1131 having an electron energy of 3 eV.

[0105] In various embodiments, the one or more transitions of the first set can comprise product ions of 676.5 m / z, product ions of 676.5, 677.5, and 678.5 m / z, product ions of 838.3, 839.3, and 840.3 m / z, or product ions of 676.5, 677.5, 678.5, 838.3, 839.3, and 840.3 m / z.

[0106] In various embodiments, the one or more transitions of the second set can comprise product ions of 292.1 m / z, product ions of 292.1, 293.1, and 294.1 m / z, product ions of 274.1, 275.1, and 276.1 m / z, or product ions of 274.1, 275.1, 276.1, 292.1, 293.1, and 294.1 m / z.

[0107] In various embodiments, in step B, processor 1140 sums the moving average of the product ion intensities of the first set at the elution times using the XICs of the first set and the second set to produce a first sum, and sums the moving average of the product ion intensities of the second set at the elution times to produce a second sum.

[0108] In various embodiments, processor 1140 also calculates the elution time of a second isomer of the one or more isomers from the second peak of the precursor XIC 1135, and performs steps B-E for the second isomer to identify one or more linkages of the sialic acid and the glycan for the second isomer.

[0109] In various embodiments, the processor 1140 calculates a first amount of the first isomer from the separation time of the first isomer and the precursor XIC 1135, and a second amount of the second isomer from the separation time of the second isomer and the precursor XIC 1135. The processor 1140 further calculates a ratio of the first amount to the second amount. The processor 1140 provides a diagnosis of the pathophysiological process based on the ratio, the one or more bonds of the sialic acid to the glycan of the identified isomer, and the one or more bonds of the sialic acid to the glycan of the identified second isomer.

[0110] 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 neurodevelopmental condition.

[0111] In various embodiments, the tandem mass spectrometer 1130 further performs, for each separation time in the plurality of separation times, a third set of one or more MRM transitions on the ion beam using the CID device 1132. An XIC is generated for each product ion of the third set. The processor 1140 further identifies, from the XICs of the third set, the one or more bonds of the sialic acid to another glycan of the isomer.

[0112] In various embodiments, the processor 1140 is configured to control or provide instructions to the separation device 1110, the ion source 1120, and the tandem mass spectrometer 1130 and analyze the collected data. The processor 1140 controls or provides instructions by, for example, controlling one or more voltage, current, or pressure sources (not shown). For example, the processor 1140 can be a separate device as shown, or can be a processor or controller of one or more devices of the tandem mass spectrometer 1130. The processor 1140 can be, but is not limited to, a controller, a computer, a microprocessor, a computer system of the Figure 11 Figure 1

[0113] Method for identifying sialic acid linkages

[0114] Figure 12 is a flowchart showing a method 1200 for identifying one or more bonds 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.

[0115] ​​In step 1210 of method 1200, 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 is separated from the sample using a separation device. The separated one or more isomers is ionized using an ion source, producing an ion beam comprising isomeric ions of precursor ions of the glycopeptide. For each separation time of a plurality of separation times, a first set and a second set of one or more MRM transitions are monitored or performed on the ion beam using an ECD device having 2-5 eV electrons. Each transition of the first set of one or more MRM transitions comprises a precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of the glycopeptide. Each transition of the second set of one or more MRM transitions comprises the precursor ion and a product ion known to be enhanced or inhibited for a second bond of the sialic acid to the glycan. A precursor XIC is generated for the precursor ion and an XIC is generated for each product ion of the first set and the second set.

[0116] In step 1220, the intensities of the product ions of the first set at the separation time are summed using the XICs of the first set and the second set using a processor, producing a first sum, and the intensities of the product ions of the second set at the separation time are summed, producing a second sum.

[0117] In step 1230, a ratio of the first sum to the second sum is calculated using a processor.

[0118] In step 1240, the ratio at the separation time is compared to a predetermined ratio range using a processor, each predetermined ratio range corresponding to a combination of one or more selections from a set of the first bond and the second bond. The one or more corresponds to one or more sialic acids known to be included in the glycopeptide.

[0119] In step 1250, the one or more bonds of the sialic acid to the glycan of the isomer are identified from the combination found to match the ratio in the comparison using a processor.

[0120] Computer program product for identifying sialic acid linkages

[0121] In various embodiments, a computer program product comprises a tangible computer readable storage medium whose contents include a program with instructions executable by a processor to perform a method for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using an ECD and MRM precursor ion to product ion transitions. The method is performed by a system comprising one or more different software modules.

[0122] Figure 13is a schematic diagram of a system 1300 including one or more different software modules that perform a method for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using ECD and MRM precursor to product ion transitions according to various embodiments. The system 1300 includes an analysis module 1310.

[0123] The analysis module 1310 calculates a separation time of an isomer of the one or more isomers of the glycopeptide of the sample from a peak of the precursor XIC. The one or more isomers are separated from the sample using a separation device. The separated one or more isomers are ionized using an ion source, producing an ion beam including isomeric ions of a precursor ion of the glycopeptide. For each separation time of a plurality of separation times, the ion beam is monitored or a first set and a second set of one or more MRM transitions are performed using an ECD device having 2-5 eV electrons. Each transition of the first set of one or more MRM transitions includes a precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of the glycopeptide. Each transition of the second set of one or more MRM transitions includes a precursor ion and a product ion known to be enhanced or inhibited for a second bond of the sialic acid to the glycan. A precursor XIC is generated for the precursor ion, and an XIC is generated for each product ion of the first set and the second set.

[0124] The analysis module 1310 sums the product ion intensities of the first set at the separation time using the XICs of the first set, producing a first sum, and sums the product ion intensities of the second set at the separation time using the XICs of the second set, producing a second sum. The analysis module 1310 calculates a ratio of the first sum to the second sum. The analysis module 1310 compares the ratio at the separation time to a predetermined ratio range, each predetermined ratio range corresponding to a combination of one or more selections from a set of the first bond and the second bond. The one or more correspond to one or more sialic acids known to be included in the glycopeptide. Finally, the analysis module 1310 identifies the one or more bonds of the sialic acid to the glycan of the isomer from the combination found matching the ratio in the comparison.

[0125] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0126] Furthermore, in describing various embodiments, the specification can have presented the method and / or process as a particular sequence of steps. However, to the extent that the method or process depends on the performance of certain steps as described, it is contemplated that the method or process can be performed with departure from the specific order set forth in this specification. For example, other steps can be performed in place of or in addition to those described herein. Still other steps can be performed prior to or after those described herein. And the method and / or process described herein can be implemented with respect to a system having more, fewer or only a single processing device. Therefore, the particular order of the steps presented is not a limitation. The steps can be executed in any order and not necessarily by the order in which they are presented.

Claims

1. A system for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using electron capture dissociation and multiple reaction monitoring precursor ion to product ion transitions, the system comprising: a separation device that separates one or more isomers of a glycopeptide from a sample; an ion source that ionizes the separated one or more isomers, producing an ion beam comprising isomeric ions of a precursor ion of a glycopeptide; a tandem mass spectrometer that, for each of a plurality of isolation times, performs a first set of one or more multiple reaction monitoring transitions and a second set of one or more multiple reaction monitoring transitions on the ion beam using an electron capture dissociation device having 2-5 eV electrons, producing an extracted ion chromatogram of the precursor ion and an extracted ion chromatogram of each product ion of the first set and the second set, each transition of the first set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of a glycopeptide, each transition of the second set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a second bond of a sialic acid to a glycan; and a processor in communication with the tandem mass spectrometer and performing the following operations: a. calculating an isolation time of a first isomer of the separated one or more isomers from a first peak of the extracted ion chromatogram of the precursor ion, b. summing product ion intensities of the first set at the isolation time using the extracted ion chromatograms of the first set and the second set, producing a first sum, and summing product ion intensities of the second set at the isolation time, producing a second sum, c. calculating a ratio of the first sum to the second sum, d. comparing the ratio at the isolation time to a predetermined ratio range, each predetermined ratio range corresponding to a combination of one or more selections from the set of the first bond and the second bond, wherein the one or more correspond to one or more sialic acids known to be included in the glycopeptide, and e. identifying one or more bonds of a sialic acid to a glycan of the isomer from the combination found in the comparison that matches the ratio.

2. The system of claim 1, wherein, The sialic acid comprises N-acetylneuraminic acid.

3. The system of claim 2, wherein, The glycan comprises galactose.

4. The system of claim 3, wherein, The first bond comprises Neu5Ac-alpha(2,6) to Gal and the second bond comprises Neu5Ac-alpha(2,3) to Gal.

5. The system of claim 1, wherein, The sialic acid comprises N-glycolylneuraminic acid.

6. The system of claim 2, wherein, The glycan comprises mannose.

7. The system of claim 1, wherein, The tandem mass spectrometer fragments the precursor ion of the first set of transitions and the second set of transitions using an electron capture dissociation device having 3 eV electrons.

8. The system of claim 1, wherein, The first set comprises one or more multiple reaction monitoring transitions comprising a product ion of 676.5 m / z.

9. The system of claim 1, wherein, The first group includes one or more multiple reaction monitoring transitions including product ions at 676.5, 677.5, and 678.5 m / z.

10. The system of claim 1, wherein, The first group includes one or more multiple reaction monitoring transitions including product ions at 838.3, 839.3, and 840.3 m / z.

11. The system of claim 1, wherein, The first group includes one or more multiple reaction monitoring transitions including product ions at 676.5, 677.5, 678.5, 838.3, 839.3, and 840.3 m / z.

12. The system of claim 1, wherein, The second group includes one or more multiple reaction monitoring transitions including product ions at 292.1 m / z.

13. The system of claim 1, wherein, The second group includes one or more multiple reaction monitoring transitions including product ions at 292.1, 293.1, and 294.1 m / z.

14. The system of claim 1, wherein, The second group includes one or more multiple reaction monitoring transitions including product ions at 274.1, 275.1, and 276.1 m / z.

15. The system of claim 1, wherein, The second group includes one or more multiple reaction monitoring transitions including product ions at 274.1, 275.1, 276.1, 292.1, 293.1, and 294.1 m / z.

16. The system of claim 1, wherein, In step b, the processor sums moving averages of product ion intensities of the first group at separation times using extracted ion chromatograms of the first group and the second group to produce the first sum and sums moving averages of product ion intensities of the second group at separation times to produce the second sum.

17. The system of claim 1, wherein, The processor further calculates a separation time of a second isomer of the one or more isomers from a second peak of the extracted ion chromatogram of the precursor ion and performs steps b-e for the second isomer to identify one or more bonds of sialic acid to glycans of the second isomer.

18. The system of claim 17, wherein, The processor further calculates a first quantity of the first isomer from the separation time of the first isomer and the extracted ion chromatogram of the precursor ion and a second quantity of the second isomer from the separation time of the second isomer and the extracted ion chromatogram of the precursor ion.

19. The system of claim 18, wherein, The processor further calculates a ratio of the first quantity to the second quantity.

20. The system of claim 1, wherein, For each of a plurality of times, the tandem mass spectrometer further performs a third set of one or more multiple reaction monitoring transitions on the ion beam using a collision induced dissociation device to produce an extracted ion chromatogram of each product ion of the third set, and wherein the processor further identifies one or more bonds of sialic acid to another glycan of an isomer from the extracted ion chromatograms of the third set.

21. A method for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using electron capture dissociation and multiple reaction monitoring precursor ion to product ion transitions, the method comprising: computing, using a processor, a separation time of an isomer of one or more isomers of a glycopeptide of a sample from peaks of an extracted ion chromatogram of a precursor ion, wherein the one or more isomers are separated from the sample using a separation device, the one or more isomers that are separated are ionized using an ion source, thereby producing an ion beam comprising isomeric ions of a precursor ion of a glycopeptide, and for each separation time of a plurality of separation times, performing, using an electron capture dissociation device having 2-5 eV electrons, a first set of one or more multiple reaction monitoring transitions and a second set of one or more multiple reaction monitoring transitions on the ion beam, thereby producing an extracted ion chromatogram of the precursor ion and an extracted ion chromatogram of each product ion of the first set and the second set, each transition of the first set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of a glycopeptide, each transition of the second set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a second bond of a sialic acid to a glycan; summing, using the processor, product ion intensities of the first set at the separation time using the extracted ion chromatograms of the first set and the second set, thereby producing a first sum, and summing product ion intensities of the second set at the separation time, thereby producing a second sum; computing, using the processor, a ratio of the first sum to the second sum; comparing, using the processor, the ratio at the separation time to a predetermined ratio range, each predetermined ratio range corresponding to a combination of one or more selections from the set of the first bond and the second bond, wherein the one or more correspond to one or more sialic acids known to be included in the glycopeptide; and identifying, using the processor, the one or more bonds of a sialic acid to a glycan of the isomer from the combination found to match the ratio in the comparison.

22. A computer program product, the computer program product comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions executable by a processor to perform a method for identifying one or more bonds of a sialic acid to a glycan of an isomer of a glycopeptide using electron capture dissociation and multiple reaction monitoring precursor ion to product ion transitions, the method comprising: providing a system, wherein the system comprises one or more different software modules, and wherein the different software modules comprise an analysis module; computing, using the analysis module, a separation time of an isomer of one or more isomers of a glycopeptide of a sample from peaks of an extracted ion chromatogram of a precursor ion, wherein the one or more isomers are separated from the sample using a separation device, the one or more isomers that are separated are ionized using an ion source, thereby producing an ion beam comprising isomeric ions of a precursor ion of a glycopeptide, and for each separation time of a plurality of separation times, performing, using an electron capture dissociation device having 2-5 eV electrons, a first set of one or more multiple reaction monitoring transitions and a second set of one or more multiple reaction monitoring transitions on the ion beam, thereby producing an extracted ion chromatogram of the precursor ion and an extracted ion chromatogram of each product ion of the first set and the second set, each transition of the first set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of a glycopeptide, each transition of the second set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a second bond of a sialic acid to a glycan; summing, using the processor, product ion intensities of the first set at the separation time using the extracted ion chromatograms of the first set and the second set, thereby producing a first sum, and summing product ion intensities of the second set at the separation time, thereby producing a second sum; computing, using the processor, a ratio of the first sum to the second sum; comparing, using the processor, the ratio at the separation time to a predetermined ratio range, each predetermined ratio range corresponding to a combination of one or more selections from the set of the first bond and the second bond, wherein the one or more correspond to one or more sialic acids known to be included in the glycopeptide; and identifying, using the processor, the one or more bonds of a sialic acid to a glycan of the isomer from the combination found to match the ratio in the comparison. The one or more isomers isolated are ionized using an ion source, producing an ion beam comprising isomeric ions of a precursor ion of a glycopeptide, and For each separation time of a plurality of separation times, performing a first set of one or more multiple reaction monitoring transitions and a second set of one or more multiple reaction monitoring transitions on the ion beam using an electron capture dissociation device having 2-5 eV electrons, producing extracted ion chromatograms of the precursor ion and of each product ion of the first set and the second set, each transition of the first set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a first bond of a sialic acid to a glycan of a glycopeptide, each transition of the second set of one or more multiple reaction monitoring transitions comprising the precursor ion and a product ion known to be enhanced or inhibited for a second bond of a sialic acid to a glycan; summing, using the analysis module, intensities of product ions of the first set at separation times using the extracted ion chromatograms of the first set and the second set, producing a first sum, and summing intensities of product ions of the second set at separation times using the extracted ion chromatograms of the first set and the second set, producing a second sum; calculating, using the analysis module, a ratio of the first sum to the second sum; comparing, using the analysis module, the ratio at separation times to a predetermined range of ratios, each predetermined range of ratios corresponding to a combination of one or more selections from the set of the first bond and the second bond, wherein one or more corresponds to one or more sialic acids known to be included in the glycopeptide; and identifying, using the analysis module, one or more bonds of a sialic acid to a glycan of an isomer from combinations found in the comparison to match the ratio.

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