WO2024171110A1

WO2024171110A1 - Glycan linkage isomer differentiation by electron activated dissociation (ead)

Info

Publication number: WO2024171110A1
Application number: PCT/IB2024/051444
Authority: WO
Inventors: Zhengwei Chen; Dilipkumar Reddy KANDULA; Lei XIONG
Original assignee: DH Technologies Development Pte Ltd
Current assignee: DH Technologies Development Pte Ltd
Priority date: 2023-02-16
Filing date: 2024-02-15
Publication date: 2024-08-22
Anticipated expiration: 2025-08-16

Abstract

Systems and methods are disclosed for determining an isomer of a non¬ derivatized glycan. A first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using electron-based dissociation (ExD) mass spectrometry are received. The isomer is determined by differentiating between a first isomer and a second isomer of the non-derivatized glycan. Differentiating between the first isomer and the second isomer includes comparing an intensity ratio of the first intensity and the second intensity to a ratio threshold value. The ratio threshold value differentiating the first isomer from the second isomer varies with the kinetic energy of the mass spectrometry The electron kinetic energy of the ExD is greater than or equal to 11 eV and less than or equal to 25 eV.

Description

GLYCAN LINKAGE ISOMER DIFFERENTIATION BY ELECTRON ACTIVATED DISSOCIATION (EAD)

RELATED APPLICATIONS

This PCT application claims the benefit of U.S. Provisional Application No. 63/485,313 filed February 16, 2023 and entitled “Glycan Linkage Isomer Differentiation by Electron Activated Dissociation (EAD),” which is incorporated herein by reference in its entirety.

SPECIFICATION

BACKGROUND

FIELD

The teachings herein relate to determining an isomer of a non-derivatized glycan.

INTRODUCTION

Glycan Linkage Background

Differences in linkages in glycoconjugates may play a crucial role in their biologic activity. For example, isomerism in linkages of sialic acid and galactose units may play such role. Sialylated glycoconjugates, for example, 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, for example, a2,6 linkages such as Neu5Ac-alpha (2,6) galactose (Gal). The linkages also include, for example, a2,3 linkages, such as Neu5Ac-alpha (2,3) Gal.

Differentiating a2,6 and a2,3 isomers of a glycan are important in diagnosing the pathophysiological processes just described. For example, in inflammatory joint disease there is a shift from a-2,6 to a-2,3 sialylation, in prostate cancer the expression of a-2,3- linked sialic acids in serum is increased, and in colorectal cancer the downregulation and upregulation of a-2,6-linked sialic acids are found in tumor tissue.

The stereochemistry structural identification in N-glycans or N-gly copeptides of a2,6 and a2,3 is conventionally reported by separation techniques, such as capillary electrophoresis (CE) and ion mobility (IMS) coupled to mass spectrometry (MS). Generally, 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 (derivatization to e.g., methylated, esterified, labeled with tags, and/or other derivatized glycans) before MS, among other differences. Thus, there remains a need for methods of glycan analysis that do not rely on chemical conversion/derivatization of glycans during sample preparation for MS analysis and, instead, use non-derivatized glycans.

For example, “High-throughput profiling of protein N-glycosylation by MALDI- TOF-MS employing linkage-specific sialic acid esterification” by Reiding et al. (describes a method based on use of carboxylic acid activators in ethanol to achieve ethyl esterification and lactonization ethyl esterification).

In another example, “collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides” by Zhao et al., describes subjecting glycans to perm ethylation prior to analysis.

As a result, additional systems and methods are needed for determining isomers of glycans in general.

Background on Mass Spectrometry Techniques

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, 1 second to 100 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/MS ^ALL. In an MS/MS ^ALL method, a precursor ion mass selection window of about 1 amu is scanned or stepped across an entire mass range. A product ion spectrum is produced for each 1 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.

Fragmentation Techniques Background 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, electron- induced dissociation (EID), electron impact excitation in organics (EIEIO), ECD, EAD, or electron transfer dissociation (EID). 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.

SUMMARY

The teachings herein relate to systems and methods for receiving intensities of a first and second product ion of non-derivatized glycan using electron-based dissociation (ExD) mass spectrometry and determining an isomer of the non-derivatized glycan from the intensities. Electron activated dissociation (EAD) is a form of ExD. 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.

A system, method, and computer program product are disclosed for determining an isomer of a non-derivatized glycan. A first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using ExD mass spectrometry are received. An isomer of the non-derivatized glycan is determined from the first intensity and the second intensity.

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

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

Figure 1 is a block diagram that illustrates a computer system, upon which embodiments of the present teachings may be implemented.

Figure 2 is an exemplary diagram showing an isomer of a glycan that includes an a2,3 unit and another isomer of the glycan that includes an a2,6 unit, in accordance with various embodiments.

Figure 3 is an exemplary diagram showing an isomer of a glycan that includes a 01,3 unit and another isomer of the glycan that includes a 01,6 unit, in accordance with various embodiments.

Figure 4 is an exemplary plot showing a mass range of a mass spectrum for the a2,3 isomer of a glycan and the same mass range of a mass spectrum for the a2,6 isomer of the glycan shown juxtaposed on either side of the x-axis where both isomers were fragmented using CID, in accordance with various embodiments.

Figure 4a is an exemplary plot showing a mass spectrum for the a2,3 isomer of a glycan and a mass spectrum for the a2,6 isomers and highlighting different mass ranges where differences were observed, in accordance with various embodiments. Figure 5 is an exemplary plot showing a mass range of a mass spectrum for the a2,3 isomer of a glycan and the same mass range of a mass spectrum for the a2,6 isomer of the glycan shown juxtaposed on either side of the x-axis where both isomers were fragmented using ExD, in accordance with various embodiments.

Figure 5 A exemplifies that dependency of Risomer parameter as a function of the different kinetic energies applied in the ExD for the a2,3 isomer and the a2,6 isomer, in accordance with various embodiments.

Figure 6 is an exemplary plot of the ratio of the intensity of the product ion at 366 m/z with respect to the intensity of the product ion at 364 m/z plotted as a function of the different kinetic energies applied in the ExD for the a2,3 isomer and the a2,6 isomer, in accordance with various embodiments.

Figure 6A exemplifies the robustness of the method in accordance with various embodiments.

Figure 6B exemplifies the application of the method in accordance to some embodiments, as applied to other glycans with a2, 3 -linkages.

Figure 7 is an exemplary diagram showing how the two different product ions of Figure 5 are produced using ExD, in accordance with various embodiments.

Figure 8 is an exemplary plot showing a mass range of a mass spectrum for the 01,3 isomer of a glycan and the same mass range of a mass spectrum for the 01,6 isomer of the glycan shown juxtaposed on either side of the x-axis where both isomers were fragmented using ExD, in accordance with various embodiments. Figure 8A is an exemplary plot showing a mass spectrum for the 01,3 isomer of a glycan and a mass spectrum for the 01,6 isomers and highlighting different mass ranges where differences were observed, in accordance with various embodiments.

Figure 9 is an exemplary plot of the ratio of the intensity of the product ion at 204 m/z with respect to the intensity of the product ion at 202 m/z plotted as a function of the different kinetic energies applied in the ExD for the 01,3 isomer and the 01,6 isomer, in accordance with various embodiments.

Figure 9A exemplifies the application of the method in accordance to some embodiments, as applied to other glycans with 01,3 and 01,4-linkages.

Figure 10 is a schematic diagram of a system for determining an isomer of a non- derivatized glycan, in accordance with various embodiments.

Figure 11 is a flowchart showing a method for determining an isomer of a non- derivatized glycan, in accordance with various embodiments.

Figure 12 is a schematic diagram of a system that includes one or more distinct software modules that perform a method for determining an isomer of a non-derivatized glycan, in accordance with various embodiments.

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. Also, 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 OF DRAWINGS

COMPUTER-IMPLEMENTED SYSTEM

Figure 1 is a block diagram that illustrates a computer system 100, upon which embodiments of the present teachings may be implemented. 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. Computer system 100 also includes a memory 106, which can be a random-access memory (RAM) or other dynamic storage device, coupled to bus 102 for storing instructions to be executed by processor 104. Memory 106 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 104. 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.

Computer system 100 may 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 controlling cursor movement on display 112. 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 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 may 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 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 100 can be connected to one or more other computer systems, like 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 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 104 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 110. Volatile media includes dynamic memory, such as memory 106. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102.

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 104 for execution. For example, the instructions may initially be carried on the 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 may optionally be stored on storage device 110 either before or after execution by processor 104.

In accordance with 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, a 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 instructions configured to be executed.

The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-obj ectoriented programming systems.

IDENTIFYING ISOMERS FROM DIAGNOSTIC PRODUCT ION INTENSITIES

As described above, sialylated glycoconjugates, for example, are known to play key roles in several pathophysiological processes including viral infection, embryogenesis, inflammation, cardiovascular diseases, cancer, and neural development. Determining the relative abundances of a2,6 and a2,3 are important in diagnosing these pathophysiological processes.

Previously, biochemical identifications identifying relative abundances of a2,6 and a2,3 have employed 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).

Figure 2 is an exemplary diagram 200 showing an isomer of a glycan that includes an a2,3 unit and another isomer of the glycan that includes an a2,6 unit, in accordance with various embodiments. Note that the ’095 Publication described a2,3 and a2,6 units as linkages. Isomer 210 includes the a2,3 unit, and isomer 220 includes the a2,6 unit. Hereinafter, isomer 210 is referred to as an a2,3 isomer and isomer 220 is referred to as an a2,6 isomer. Glycan isomers can have other differing units.

Figure 3 is an exemplary diagram 300 showing an isomer of a glycan that includes a 01,3 unit and another isomer of the glycan that includes a 01,6 unit, in accordance with various embodiments. Isomer 310 includes the 01,3 unit, and isomer 320 includes the 01,6 unit. Hereinafter, isomer 310 is referred to as a 01,3 isomer and isomer 320 is referred to as a 01,6 isomer.

In order to differentiate the isomers of a glycan using MS/MS, diagnostic ions must be found. Conventionally, MS/MS is performed using CID.

Figure 4 is an exemplary plot 400 showing a mass range of a mass spectrum for the a2,3 isomer of a glycan and the same mass range of a mass spectrum for the a2,6 isomer of the glycan shown juxtaposed on either side of the x-axis where both isomers were fragmented using CID, in accordance with various embodiments. In plot 400, product ion peak 410 shows the intensity of the product ion at 366 m/z for the a2,3 isomer. Peak 420 shows the intensity of the product ion at 366 m/z for the a2,6 isomer. Although peak 410 and 420 have slightly different intensities, it is not possible to differentiate the a2,3 isomer and the a2,6 isomer using these product ions. As a result, Figure 4 shows that it is not possible to differentiate the a2,3 isomer and the a2,6 isomer using CID.

Figure 4a is an exemplary plot showing a mass spectrum for the a2,3 isomer of a glycan and a mass spectrum for the a2,6 isomers and highlighting different mass ranges where differences were observed, in accordance with various embodiments. The range identified with a check mark was found to be diagnostic for the differentiation between the isomers.

Figure 5 is an exemplary plot 500 showing a mass range of a mass spectrum for the a2,3 isomer of a glycan and the same mass range of a mass spectrum for the a2,6 isomer of the glycan shown juxtaposed on either side of the x-axis where both isomers were fragmented using ExD, in accordance with various embodiments. More specifically, the isomers of Figure 5 were fragmented using the ExD method of a ZenoTOF 7600 mass spectrometer from SCIEX of Framingham, MA. In plot 500, product ion peak 511 shows the intensity of the product ion at 364 m/z and product ion peak 512 shows the intensity of the product ion at 366 m/z for the a2,3 isomer. Product ion peak 521 shows the intensity of the product ion at 364 m/z and product ion peak 522 shows the intensity of the product ion at 366 m/z for the a2,6 isomer.

Plot 500 shows that the intensity ratio of these two product ions of the glycan is different for the two different isomers. As a result, Figure 5 shows that ExD can produce diagnostic ions for differentiating glycan isomers. In order to determine if this holds true for different levels of kinetic energy applied in the ExD, the intensity ratio of these two product ions was measured using different kinetic energies. Figure 5 A exemplifies that dependency of Risomer score as a function of the different kinetic energies applied in the ExD for the a2,3 isomer and the a2,6 isomer, in accordance with various embodiments. Risomer score is calculated as (the ratio of the intensity of the product ion at 366 m/z with respect to the intensity of the product ion at 364 m/z for a2,6 isomer) divided by (the ratio of the intensity of the product ion at 366 m/z with respect to the intensity of the product ion at 364 m/z for a2,3 isomer). The Risomer score is an indicator of the differentiation between the a2,3 isomer and the a2,6 isomer. As the Risomer score increases, the differentiation between the two isomers improves. The correlation between the Risomer score and the KE value has been established, with higher KE values providing better differentiation. Consequently, the KE values in the higher range of 11-25 eV were selected for analysis.

Figure 6 is an exemplary plot 600 of the ratio of the intensity of the product ion at 366 m/z with respect to the intensity of the product ion at 364 m/z plotted as a function of the different kinetic energies applied in the ExD for the a2,3 isomer and the a2,6 isomer, in accordance with various embodiments. Points 610 show the intensity ratio for the a2,3 isomer. Points 620 show the intensity ratio for the a2,6 isomer. Plot 600 shows the intensity ratio of these two product ions is consistently different for the two different isomers for a range of kinetic energies from at least 11 eV to 25 eV. Thus, Figure 6 shows that these two product ions can be used to differentiate glycan isomers using ExD for a kinetic energy range of at least 11 eV to 25 eV.

Figure 6A exemplifies the robustness of the method in accordance with various embodiments. It further exemplifies an exemplary plot of the ratio of the intensity of the product ion at 366 m/z with respect to the intensity of the product ion at 364 m/z plotted as a function of the different kinetic energies applied in the ExD for the a2,3 isomer and the a2,6 isomer, in accordance with various embodiments -performed in triplicate.

Figure 7 is an exemplary diagram 700 showing how the two different product ions of Figure 5 are produced using ExD, in accordance with various embodiments. Fragmentation process 710 shows how ExD produces a [Y2] ion (366 m/z). Fragmentation process 720 shows how ExD produces a [Y2-2H] ion (364 m/z). Figure 7 confirms that the diagnostic ions of Figure 5 can be used for differentiating glycan isomers.

Figures 5-7 relate to the a2,3 and a2,6 isomers of a glycan. The 01,3 and 01,6 isomers of a glycan can similarly be differentiated using two product ions.

Figure 8 is an exemplary plot 800 showing a mass range of a mass spectrum for the 01,3 isomer of a glycan and the same mass range of a mass spectrum for the 01,6 isomer of the glycan shown juxtaposed on either side of the x-axis where both isomers were fragmented using ExD, in accordance with various embodiments. Again, the isomers of Figure 8 were fragmented using the ExD method of a ZenoTOF 7600 mass spectrometer from SCIEX of Framingham, MA. In plot 800, product ion peak 811 shows the intensity of the product ion at 202 m/z and product ion peak 812 shows the intensity of the product ion at 204 m/z for the 01,3 isomer. Product ion peak 821 shows the intensity of the product ion at 202 m/z and product ion peak 822 shows the intensity of the product ion at 204 m/z for the 01,6 isomer.

Plot 800 shows that the intensity ratio of these two product ions of the glycan is different for the two different isomers. In order to determine if this holds for different levels of kinetic energy applied in the ExD, the intensity ratio of these two product ions was measured using different kinetic energies.

Figure 8A is an exemplary plot showing a mass spectrum for the 01,3 isomer of a glycan and a mass spectrum for the 01,6 isomers and highlighting different mass ranges where differences were observed, in accordance with various embodiments. The range identified with a check mark was found to be diagnostic for the differentiation between the isomers.

Figure 9 is an exemplary plot 900 of the ratio of the intensity of the product ion at 204 m/z with respect to the intensity of the product ion at 202 m/z plotted as a function of the different kinetic energies applied in the ExD for the 01,3 isomer and the 01,6 isomer, in accordance with various embodiments. Points 910 show the intensity ratio for the 01,3 isomer. Points 920 show the intensity ratio for the 01,6 isomer. Plot 900 shows that the intensity ratio of these two product ions is consistently different for the two different isomers for a range of kinetic energies from at least 11 eV to 25 eV. Thus, Figure 9 shows that these two product ions can be used to differentiate glycan isomers using ExD for a kinetic energy range of at least 11 eV to 25 eV.

System for determining an isomer of a non-derivatized glycan

Figure 10 is a schematic diagram 1000 of a system for determining an isomer of a non-derivatized glycan, in accordance with various embodiments. The system of Figure 10 includes processor 1040.

Processor 1040 receives a first intensity 1035 of a first product ion and a second intensity 1036 of a second product ion for a non-derivatized glycan 1001 produced using ExD mass spectrometry. Processor 1040 determines an isomer of the non-derivatized glycan from first intensity 1035 and second intensity 1036.

In various embodiments, processor 1040 determines an isomer of the non-derivatized glycan by differentiating between a first isomer and a second isomer of the non-derivatized glycan.

In various embodiments, processor 1040 differentiates between the first isomer and the second isomer by comparing an intensity ratio of first intensity 1035 and second intensity 1036 to a ratio threshold value. As shown in Figure 6, points 620 corresponding to the intensity ratio for the a2,6 isomer are generally larger than points 610 corresponding to the intensity ratio for the a2,3 isomer. So, the ratio threshold value for differentiating the a2,6 isomer and the a2,3 isomer is between points 610 and 620. However, both points 610 and 620 are increasing in value. In addition, points 620 corresponding to the intensity ratio for the a2,6 isomer are increasing faster with the increase in kinetic energy of the mass spectrometry than points 610 corresponding to the intensity ratio for the a2,3 isomer.

In various embodiments, therefore, the ratio threshold value differentiating the first isomer from the second isomer varies with the kinetic energy of the mass spectrometry. In various embodiments, the first product ion is a [Y] ion and the second product ion is a [Y-2H] ion or the first product ion is a [Y-2H] ion and the second product ion is a [Y] ion.

In various embodiments, the first isomer is an a2,3 isomer and the second isomer is an a2,6 isomer or the first isomer is an a2,6 isomer and the second isomer is an a2,3 isomer.

In various embodiments, the first isomer is a 01,3 isomer and the second isomer is a 01,6 isomer or the first isomer is an 01,6 isomer and the second isomer is a 01,3 isomer.

In various embodiments, the non-derivatized glycan is an O-glycan or an N-glycan.

In various embodiments, the isomer includes a sialic acid unit or a galactose unit.

In various embodiments, the ExD is ECD.

In various embodiments, the electron kinetic energy of the ExD is greater than or equal to 11 eV and less than or equal to 25 eV.

In various embodiments, the system of Figure 10 further includes separation device 1010, ion source 1020, and tandem mass spectrometer 1030.

Separation device 1010 separates the non-derivatized glycan 1001 from a sample. Separation device 1010 can separate the non-derivatized glycan 1001 from the sample using any separation technique including, but not limited to, LC, CE, or IMS.

Ion source 1020 ionizes the non-derivatized glycan 1001, producing an ion beam that includes a precursor ion of the non-derivatized glycan 1001. Ion source 1020 is shown as performing electrospray ionization (ESI) (e.g., nanospray) but can be any type of ion source. Ion source 1020 is shown as part of tandem mass spectrometer 1030 but can also be a separate device. Tandem mass spectrometer 1030 includes ExD device 1031. Tandem mass spectrometer 1030 fragments the precursor ion of the non-derivatized glycan 1001 using ExD device 1031. Tandem mass spectrometer 1030 measures first intensity 1035 of the first product ion and second intensity 1036 of the second product ion for non-derivatized glycan 1001.

Processor 1040 is in communication with separation device 1010, ion source 1020, and tandem mass spectrometer 1030.

Method for determining an isomer of a non-derivatized glycan

Figure 11 is a flowchart showing a method 1100 for determining an isomer of a non- derivatized glycan, in accordance with various embodiments.

In step 1110 of method 1100, a first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using ExD mass spectrometry are received.

In step 1120, an isomer of the non-derivatized glycan is determined from the first intensity and the second intensity.

Computer Program Product for determinins an isomer of a non-derivatized glycan

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 determining an isomer of a non-derivatized 1 glycan. This method is performed by a system that includes one or more distinct software modules.

Figure 12 is a schematic diagram of a system 1200 that includes one or more distinct software modules that perform a method for determining an isomer of a non-derivatized glycan, in accordance with various embodiments. System 1200 includes an input module 1210 and an analysis module 1220.

Input module 1210 receives a first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using ExD mass spectrometry.

Analysis module 1220 determines an isomer of the non-derivatized glycan from the first intensity and the second intensity.

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 of skill in the art.

Further, in describing various embodiments, the specification may have presented a method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for determining an isomer of a non-derivatized glycan, comprising:

(a) receiving a first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using electron-based dissociation (ExD) mass spectrometry; and

(b) determining an isomer of the non-derivatized glycan from the first intensity and the second intensity.

2. The method of any combination of the preceding method claims, wherein determining an isomer comprises differentiating between a first isomer and a second isomer of the non-derivatized glycan.

3. The method of any combination of the preceding method claims, wherein differentiating between the first isomer and the second isomer comprises comparing an intensity ratio of the first intensity and the second intensity to a ratio threshold value.

4. The method of any combination of the preceding method claims, wherein the ratio threshold value differentiating the first isomer from the second isomer varies with the kinetic energy of the mass spectrometry.

5. The method of any combination of the preceding method claims, wherein the first product ion comprises a [Y] ion and the second product ion comprises a [Y-2H] ion or the first product ion comprises a [Y-2H] ion and the second product ion comprises a [Y] ion.

6. The method of any combination of the preceding method claims, wherein the first isomer comprises an a2,3 isomer and the second isomer comprises an a2,6 isomer or the first isomer comprises an a2,6 isomer and the second isomer comprises an a2,3 isomer.

7. The method of any combination of the preceding method claims, wherein the first isomer comprises a 01,3 isomer and the second isomer comprises a 01,6 isomer or the first isomer comprises an 01,6 isomer and the second isomer comprises a 01,3 isomer.

8. The method of any combination of the preceding method claims, wherein the non- derivatized glycan comprises an O-glycan.

9. The method of any combination of the preceding method claims, wherein the non- derivatized glycan comprises an N-glycan.

10. The method of any combination of the preceding method claims, wherein the isomer comprises a sialic acid unit.

11. The method of any combination of the preceding method claims, wherein the isomer comprises a galactose unit.

12. The method of any combination of the preceding method claims, wherein the ExD comprises electron capture dissociation (ECD).

13. The method of any combination of the preceding method claims, wherein an electron kinetic energy of the ExD comprises greater than or equal to 11 eV and less than or equal to 25 eV.

14. A computer program product, comprising a non-transitory tangible computer- readable storage medium whose contents cause a processor to perform a method for determining an isomer of a non-derivatized glycan, comprising: providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise an input module and an analysis module;

(a) receiving a first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using electron-based dissociation (ExD) mass spectrometry using the input module; and

(b) determining an isomer of the non-derivatized glycan from the first intensity and the second intensity using the analysis module.

15. A system for determining an isomer of a non-derivatized glycan, comprising: a processor that

(a) receives a first intensity of a first product ion and a second intensity of a second product ion for a non-derivatized glycan produced using electron-based dissociation (ExD) mass spectrometry; and

(b) determines an isomer of the non-derivatized glycan from the first intensity and the second intensity.