Method for analyzing mucin types and glycan structures of o-linked glycopeptides

Abstract

The present invention relates to a method for analyzing mucin types and glycan structures of O-linked glycopeptides. More specifically, the present invention relates to a method for identifying types of O-linked glycopeptides using a high-resolution mass spectrometer, a method for analyzing glycan binding positions of O-linked glycopeptides, and a computer-readable recording medium recording a program applied to perform the methods. The methods according to the present invention can efficiently and accurately analyze various mucin types and glycan structures of O-linked glycopeptides, and can be usefully employed to easily discover disease markers from samples for prediction or diagnosis or to analyze the structures of O-linked glycopeptides in glycoprotein drugs by using a high-resolution mass spectrometer.

Description

Method for analyzing the mucin type and sugar chain structure of O-linked glycopeptides

[0001] The present invention relates to a method for analyzing the mucin type and sugar chain structure of an O-linked glycopeptide. More specifically, the present invention relates to a method for identifying the type of an O-linked glycopeptide using a high-resolution mass spectrometer, a method for analyzing the sugar chain bonding position of an O-linked glycopeptide, and a computer-readable recording medium recording a program applicable to performing the above method.

[0002] Glycosylation plays a crucial role in regulating protein structure and function. Recent studies have also revealed a link between various diseases, such as immune disorders, and abnormal glycan structures. These findings make structural analysis of glycoproteins and glycopeptides crucial in diverse fields, including life sciences, medicine, and pharmaceutical development.

[0003] Protein glycosylation is divided into N-linked and O-linked glycosylation. N-linked glycosylation occurs in the endoplasmic reticulum (ER), whereas O-linked glycosylation occurs in the ER, Golgi apparatus, or cytoplasm. O-linked glycosylation is classified into non-mucin type and mucin type, and O-linked glycosylation that occurs in mammals is mainly mucin type. Mucin type glycosylation is complex because it is initiated primarily by the attachment of N-acetylgalactosamine (GalNAc) to serine or threonine and is enzymatically catalyzed directly without the aid of precursors such as dolichol. In addition, mucin type O-linked glycoproteins are mainly found in the cytoplasm or nucleus. However, O-linked glycosylation has been relatively less studied than N-linked glycosylation and is still not widely known (KR 10-2422169 B1).

[0004] Conventional methods used for qualitative and quantitative analysis of O-linked glycopeptides have the following limitations. First, although the general basic structure of O-linked glycopeptides is known, the composition of new O-linked glycopeptides cannot be known in advance because O-linked glycopeptides exist in a wide variety of forms. Therefore, it is difficult to create a complete database of O-linked glycopeptides. Second, the mucin type of O-linked glycopeptide is most commonly found in animals, which, as described above, begins with the attachment of N-acetylgalactosamine to the oxygen atom of serine or threonine. However, since serine and threonine are frequently found in the amino acid sequences that constitute proteins, the O-linked glycopeptide to be analyzed may have two or more O-linked glycosylation sites.

[0005] Accurate analysis of O-linked glycopeptides is essential for disease diagnosis and therapeutic development. However, due to the complex structure and diversity of these glycopeptides, existing methods using mass spectrometry spectra have limitations in accurately identifying and analyzing the types and structures of these sugars. Therefore, the development of new analytical methods to accurately identify and analyze the diverse sugar types and structures within O-linked glycopeptides is urgently needed.

[0006] Accordingly, the present inventors have endeavored to develop an efficient new analytical method for the accurate analysis of the types and sugar chain structures of O-linked glycopeptides. As a result, based on mass spectral analysis using higher-energy collisional dissociation (HCD), the ratio of GlcNAc to GalNAc (GGRatio) was calculated and specific ion (B ion and Y ion) peaks were identified in the HCD spectrum. Based on this, a novel method capable of automatically classifying various mucin types of O-linked glycopeptides and determining single or multiple O-glycosylation sites was established, thereby completing the present invention.

[0007] To achieve the above object, one aspect of the present invention provides a method for classifying O-linked glycopeptide types, including the steps of: obtaining mass spectrum data for a sample; selecting glycopeptide spectrum data using tandem spectrum (MS / MS) data extracted from the mass spectrum data; selecting O-linked glycopeptides from the selected glycopeptide spectrum data; obtaining information on GGRatio, B ion peaks, and Y ion peaks from the selected glycopeptide spectrum data; and classifying sample peptides into eight types according to conditions.

[0008] Another aspect of the present invention provides a method for analyzing the number of sugar chains in O-linked glycopeptides by analyzing dHN, pNGS and NGS conditions.

[0009] Another aspect of the present invention provides a method for analyzing O-linked glycopeptides based on the type of O-linked glycopeptide, B ion, Y ion, type of O-linked glycopeptide, and number of sugar chains in the O-linked glycopeptide.

[0010] The method according to the present invention can efficiently analyze various mucin types and sugar chain structures of O-linked glycopeptides more quickly and accurately than existing methods. Furthermore, by utilizing a high-resolution mass spectrometer, it can be useful for detecting markers for diseases, including cancer, from samples for prediction or diagnosis, or for analyzing the structures of O-linked glycopeptides in glycoprotein pharmaceuticals.

[0011] Figure 1 is a flowchart illustrating a method for analyzing the sugar type of an O-linked glycopeptide according to one embodiment of the present invention.

[0012] Figure 2 illustrates an algorithm for analyzing the number of sugar chains in an O-linked glycopeptide according to one embodiment of the present invention.

[0013] Figures 3a to 3c illustrate a specific example of analyzing an O-linked glycopeptide based on the type of sugar in the O-linked glycopeptide and the number of sugar chains in the O-linked glycopeptide.

[0014] FIG. 4 is a graph showing the intensities of HexNAc-2H2O-Acetyl (m / z 126), HexNAc-H2O-Acetyl (m / z 144), HexNAc-2H2O-CH2O (m / z 138), HexNAc-2H2O (m / z 168), Peptide+HexHexNAc (m / z 1,027), and Peptide+2HexNAc (m / z 1,041) ions for an O-linked glycopeptide according to one embodiment of the present invention under various HCD fragmentation energy conditions.

[0015] FIGS. 5a and 5b are graphs comparing the relative intensity ratios (GGRatio) of HexNAc-2H2O-CH2O (m / z 138) and HexNAc-2H2O (m / z 168) ions to HexNAc-2H2O-Acetyl (m / z 126) and HexNAc-H2O-Acetyl (m / z 144) ions for O-linked glycopeptides according to one embodiment of the present invention. Specifically, the results of classification into Core 1 type and Core 2 type of O-linked glycopeptides based on a GGRatio value of 1.81 (FIG. 5a) and the results of classification into Core 2 type and Extended Core 2 type of O-linked glycopeptides based on a GGRatio value of 4.37 (FIG. 5b) are shown.

[0016] FIG. 6a and FIG. 6b are specific examples of the results of calculating the relative intensity ratio (GGRatio) of HexNAc-2H2O-Acetyl (m / z126) and HexNAc-H2O-Acetyl (m / z144) ions to HexNAc-2H2O-CH2O (m / z138) and HexNAc-2H2O (m / z168) ions for a sample peptide of the present invention.

[0017] Figure 7 shows various sugar types and their m / z values.

[0018] Figure 8 shows the types of oxonium ions and their m / z values.

[0019] Figure 9 shows the types of glycan B ions and their m / z values.

[0020] Figures 10a to 10q classify and illustrate 80 types of O-linked sugar chains.

[0021] O-linked glycopeptide type classification method

[0022] One aspect of the present invention provides a method for classifying O-linked glycopeptide types, comprising the following steps.

[0023] Specifically, a method for classifying O-linked glycopeptide types is provided, including: 1) obtaining mass spectrum data for a sample; 2) selecting glycopeptide spectrum data using tandem spectrum (MS / MS) data extracted from the mass spectrum data; 3) selecting O-linked glycopeptides from the selected glycopeptide spectrum data; 4) obtaining information on GGRatio, B ion peaks, and Y ion peaks from the selected glycopeptide spectrum data; and 5) classifying sample peptides into eight types according to the following conditions.

[0024] At this time, if GGRatio<1.81, it can be determined to be an O-linked glycopeptide of the Core 1 type.

[0025] Additionally, if 1.81≤GGRatio<4 and there is no B ion peak for 2HexNAc (m / z 407.15) or Y ion peak for PEP+2HexNAc, it can be determined to be an O-linked glycopeptide of the Extended Core 1 type.

[0026] In addition, if 1.81≤GGRatio<4.37 and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc, but there is no Y ion peak for PEP+HexHexNAc, it can be determined to be an O-linked glycopeptide of the Core 2 type.

[0027] In addition, if GGRatio≥4.37 and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc, and a Y ion peak for PEP+HexHexNAc, it can be determined to be an O-linked glycopeptide of the Extended Core 2 type.

[0028] In addition, if GGRatio<4.37, a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc exists, a Y ion peak for PEP+HexHexNAc does not exist, and a B ion peak for 3HexNAc (m / z 610.72) does not exist, it can be judged to be an O-linked glycopeptide of the Core 3 type.

[0029] In addition, if GGRatio≥4.37, a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc exists, a Y ion peak for PEP+HexHexNAc does not exist, and a B ion peak for 3HexNAc (m / z 610.72) does not exist, it can be determined to be an O-linked glycopeptide of the Extended Core 3 type.

[0030] In addition, if GGRatio<4.37, a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc exists, a Y ion peak for PEP+HexHexNAc does not exist, and a B ion peak for 3HexNAc (m / z 610.72) exists, it can be determined to be an O-linked glycopeptide of the Core 4 type.

[0031] In addition, if GGRatio≥4.37, a B ion peak for 2HexNAc or a Y ion peak for PEP+2HexNAc exists, a Y ion peak for PEP+HexHexNAc does not exist, and a B ion peak for 3HexNAc (m / z 610.72) exists, it can be judged to be an O-linked glycopeptide of the Extended Core 4 type.

[0032] Each step of the above O-linked glycopeptide type classification method can be performed as follows.

[0033] First, it may include a step of acquiring mass spectral data for the sample.

[0034] The term "sample" as used herein may be a protein or peptide containing a glycopeptide. Specifically, the sample may be derived from animals, including humans, plants, insects, yeast, etc. The sample is not particularly limited as long as it is a sample likely to contain a glycopeptide. For example, a sample containing a glycoprotein may be subjected to glycoprotein fragmentation treatment. Furthermore, glycopeptides may be concentrated from the obtained peptides through hydrophilic interaction chromatography. At this time, the sample containing a glycoprotein may be a biological sample or an environmental sample. For example, biological samples such as whole blood, serum, plasma, urine, saliva, cerebrospinal fluid, cells and cell cultures, and tissues may be exemplified. The biological sample may be unpurified or may contain a glycoprotein purified according to a known technique. Furthermore, the sample may be subjected to treatments such as defatting, desalting, and protein fractionation.

[0035] As used herein, the term "glycosylation" refers to the covalent attachment of a sugar to a protein surface, wherein the sugar is linked to a serine or threonine residue to form an O-linked glycan (O-glycan). The most common group of O-linked glycans are GalNAc O-glycans, which are linked to serine (Ser) or threonine (Thr) by N-acetyl-galactosamine (GalNAc), and can be joined to additional monosaccharides. Therefore, the glycosylation sites of an O-linked peptide can be calculated based on the number of serine (Ser) and threonine (Thr) residues within the peptide.

[0036] O-linked GalNAc glycans can always have an α-linked N-acetylgalactosamine residue attached to serine or threonine. GalNAc can be linked to residues such as fucose (Fuc), galactose (Gal), glucosamine (Glc), mannose (Man), and N-acetylglucosamine (GlcNAc).

[0037] The term "hydrolysis" as used herein may refer to a process of fragmenting glycoproteins, separating only sugars from the glycoprotein. The hydrolysis may be performed using any method well known in the art. For this purpose, treatment may be performed with any enzyme selected from the group consisting of trypsin, chymotrypsin, and proteinase K.

[0038] Tandem spectral data can be obtained from mass spectral data.

[0039] The mass spectrum can be obtained by analyzing a polypeptide obtained by hydrolyzing a glycoprotein in a sample using a high-resolution mass spectrometer. Specifically, a mass spectrometer can be used to efficiently qualitatively and quantitatively analyze glycopeptides, such as O-linked glycopeptides, which are complex, diverse, and present in a low concentration in a sample compared to general peptides. The results obtained from the mass spectrometer can be used to identify glycopeptides using an M-score (or MM-score), and quantitatively analyze the identified glycopeptides. The mass spectrometer can have a mass resolution of 10,000 or more and a mass accuracy of 50 ppm or less. In one embodiment, the mass spectrometer is an Orbitrap. TM Mass spectrometer and / or Q Exactive TMIt could be a mass spectrometer.

[0040] As used herein, the term "tandem spectrum (MS / MS)" refers to a spectrum analyzed by selecting ions of interest or ions with relatively high sensitivity from the entire mass spectrum (MS). Tandem mass spectrometry can be performed by analyzing the mass of the tandem spectrum. The tandem spectrum may be a high energy collision dissociation-MS / MS (HCD-MS / MS) spectrum.

[0041] Second, it may include a step of selecting peptide spectral data using tandem spectrum (MS / MS) data extracted from mass spectral data.

[0042] At this time, the selection of the peptide spectrum data can be performed by calculating the M-score from the tandem spectrum (MS / MS) data according to the following [Mathematical Formula 1];

[0043] [Mathematical Formula 1]

[0044]

[0045] Here, And,

[0046] N is the total number of multiple oxonium ion peaks,

[0047] n is the number of oxonium ions identified in the spectral data of the sample,

[0048] Ii is the intensity of the observed ith peak,

[0049] Imax is the base peak intensity (BPI),

[0050] C is a constant value.

[0051] In addition, the plurality of oxonium ion peaks are as described in the table below.

[0052]

[0053] Third, a step of selecting O-linked glycopeptides from the selected glycopeptide spectrum data may be included.

[0054] O-linked glycopeptides can be obtained using the oxonium ion peak values ​​described above. The m / z values ​​for the sugar chains and glycopeptides are as described in Figures 7 to 9.

[0055] Fourth, it may include a step of obtaining information on GGRatio, B ion peak and Y ion peak from the selected peptide spectrum data.

[0056] The term "GGRatio" as used herein refers to a calculated value of the ratio of N-acetylglucosamine (GlcNAc) to N-acetylgalactosamine (GalNAc). Specifically, it can be derived by analyzing the relative intensity ratio of HexNAc-2H2O-Acetyl (m / z 126) and HexNAc-H2O-Acetyl (m / z 144) ions to HexNAc-2H2O-CH2O (m / z 138) and HexNAc-2H2O (m / z 168) ions in tandem spectrum (MS / MS) data.

[0057] Specifically, the above GGRatio can be obtained by the following [Mathematical Formula 2]:

[0058] [Equation 2]

[0059]

[0060] Here, And,

[0061] 138, 168, 126 and 144 represent the mass-to-charge ratio (m / z) for specific peaks,

[0062] Ii is the intensity of the observed ith peak,

[0063] Imax is base peak intensity (BPI).

[0064] The term "B ion" as used herein refers to an ion generated from a sugar chain. The above oxonium ions are representative examples, and the m / z values ​​of these ions can be used to identify part of the sugar chain sequence. Specifically, the B ion m / z is as shown in Figure 9.

[0065] As used herein, the term "Y ion" refers to an ion occurring in the sequence of a sugar chain and a peptide chain. In particular, Y-ions can be used to analyze sugar chains, including the amino acid sequence of a peptide.

[0066] Fifth, it may include a step of classifying the sample peptides into eight types according to the following conditions.

[0067] At this time, the sample peptides can be divided into eight types, such as i) Core 1 type O-linked glycopeptide, ii) Extended Core 1 type O-linked glycopeptide, iii) Core 2 type O-linked glycopeptide, iv) Extended Core 2 type O-linked glycopeptide, v) Core 3 type O-linked glycopeptide, vi) Extended Core 3 type O-linked glycopeptide, vii) Core 4 type O-linked glycopeptide, and viii) Extended Core 4 type O-linked glycopeptide.

[0068] Additionally, the above eight types of O-linked glycopeptides can be defined as having the following GlcNAc number and HexNAc number:

[0069] O-linked glycopeptides of Core 1 type have GlcNAc=0 ​​and HexNAc=1,

[0070] O-linked glycopeptides of the Extended Core 1 type are GlcNAc≥1 and HexNAc=2,

[0071] O-linked glycopeptides of the Core 2 type are GlcNAc=1 and HexNAc=2.

[0072] O-linked glycopeptides of the Extended Core 2 type are GlcNAc≥2 and HexNAc=3,

[0073] Core 3 type O-linked glycopeptides are GlcNAc=1 and HexNAc=2.

[0074] O-linked glycopeptides of the Extended Core 3 type are GlcNAc≥2 and HexNAc=3,

[0075] Core 4 type O-linked glycopeptides are GlcNAc=2, HexNAc=3, and

[0076] O-linked glycopeptides of the Extended Core 4 type are GlcNAc≥3 and HexNAc=4.

[0077] Method for analyzing the number of sugar chains in O-linked glycopeptides

[0078] Another aspect of the present invention provides a method for analyzing the number of sugar chains in an O-linked glycopeptide, comprising the following steps.

[0079] Specifically, a method for analyzing the number of sugar chains in an O-linked glycopeptide is provided, including: 1) a step of determining that there is one glycosylation site if the condition dHN=0 is satisfied; and 2) a step of determining that there are multiple glycosylation sites if the condition pNGS≤NGS is satisfied.

[0080] At this time, the above dHN is the number of HexNAc derived according to the following [Mathematical Formula 3]:

[0081] [Equation 3]

[0082] dHN = nHN - pHN.

[0083] Additionally, the above nHN represents the number of HexNAc of O-glycopeptides observed in the sample.

[0084] Additionally, the pHN represents the number of HexNAc predicted according to the following conditions (i) to (iv):

[0085] (i) If GGRatio≤1.8, HexNAc is 1,

[0086] (ii) 1.81 <GGRatio≤4.37 인 경우, HexNAc는 2이며,

[0087] (iii) 4.37 <GGRatio≤7.0 인 경우, HexNAc는 3이며,

[0088] (iv) 7.0 <GGRatio인 경우, HexNAc는 4이며.

[0089] Additionally, the above pNGS represents the number of observable O-glycosylation sites derived according to the following [Mathematical Formula 4]:

[0090] [Equation 4]

[0091] pNGS = dHN + 1.

[0092] Additionally, the above NGS indicates the number of O-glycosylation sites in the non-glycosylated sample.

[0093] As a specific example, TPLPPTSAHGNVAEGETKPDPDVTER (SEQ ID NO: 1) may have five O-glycosylation sites at threonine (T) and serine (S) positions as shown.

[0094] O-linked glycopeptide analysis method

[0095] Another aspect of the present invention provides a method for analyzing O-linked glycopeptides comprising the following steps.

[0096] Specifically, the method includes a step of deriving B ion, Y ion, and O-linked glycopeptide types from the above-described method; a step of deriving the number of sugar chains in the O-linked glycopeptide from the above-described method; and a step of deriving the type of O-linked glycopeptide in [Table 2] based on the O-linked glycopeptide type, B ion type, and Y ion type.

[0097] First, it may include a step of deriving B ion, Y ion and O-linked glycopeptide types.

[0098] The above B ion, Y ion and O-linked glycopeptide types and methods for deriving them are as described.

[0099] Second, it may include a step of deriving the number of sugar chains in the O-linked glycopeptide.

[0100] The number of sugar chains in an O-linked glycopeptide and the method for deriving the same are as described above.

[0101] Third, it may include a step of analyzing O-linked glycopeptides based on O-linked glycopeptide type, B ion, and Y ion.

[0102] Referring to the types of B ions and Y ions described above and Figure 9, the sugar chain pattern of the sample can be classified as shown in Table 2 below.

[0103]

[0104]

[0105]

[0106] The sugar chain types and structures of Table 2 above are shown more specifically in Figures 10a to 10q.

[0107] O-linked glycopeptide computer-readable recording medium

[0108] Another aspect of the present invention provides a computer-readable recording medium recording a program applied to perform the above-described O-linked glycopeptide type classification method; a program applied to perform the above-described method for analyzing the number of sugar chains in an O-linked glycopeptide; and / or a program applied to perform the above-described method for analyzing an O-linked glycopeptide.

[0109] The above “method for identifying O-linked glycopeptide type” and “method for analyzing sugar chain bonding position of O-linked glycopeptide” are as described above.

[0110] The above methods may be implemented at least partially in the form of program instructions and recorded on a computer-readable recording medium. For example, they may be implemented with a program product comprising a computer-readable medium containing program code, which may be executed by a processor to perform any or all of the steps, operations, or processes described.

[0111] The computer is a computing device having one or more alternative and special-purpose processors, memory, storage, and networking components (either wireless or wired). The computer may run an operating system compatible with Microsoft Windows, such as a Linux distribution.

[0112] The above program instruction form may be collectively referred to as software, which may include a computer program, code, instructions, or a combination of one or more of these, and may configure a processing device to operate as desired or independently or collectively command the processing device. The software and / or data may be permanently or temporarily embodied in any type of machine, component, physical device, virtual equipment, computer storage medium, or device to be interpreted by the processing device or to provide instructions or data to the processing device.

[0113] The computer-readable recording medium may include program commands, data files, data structures, etc., either singly or in combination. The program commands recorded on the medium may be specially designed and configured for the implementation example, or may be known and available to those skilled in the art of computer software. Examples of the computer-readable recording medium include magnetic media such as hard disks, optical media such as CD-ROMs and DVDs, magneto-optical media such as floptical disks, and hardware devices specially configured to store and execute program commands, such as ROMs, RAMs, and flash memories. Examples of program commands include not only machine language codes generated by a compiler, but also high-level language codes that can be executed by a computer using an interpreter, etc. The hardware devices may be configured to operate as one or more software modules to perform the operations of the implementation example.

[0114] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0115] Example 1. Analysis of human serum samples

[0116] Example 1.1. Preparation of human serum samples

[0117] Human serum samples were purchased from Sigma Alderrich. The purchased serum samples were suspended in 100 μl of 50 mM ammonium bicarbonate. The suspension was reacted at 95°C to initially denature the protein. Additionally, 5 μl of 200 mM dithiothreitol was added and reacted at 60°C for 1 hour to cleave disulfide bonds. Next, 10 μl of 200 mM iodoacetic acid was added to alkylate the cleaved disulfide bonds and reacted in the dark for 1 hour. Finally, 10 μg of trypsin was added to the denatured protein and hydrolyzed at 37°C for 16 hours to fragment it into peptides. The hydrolyzed sample was concentrated using a ZIC®-HILIC column and prepared.

[0118] Example 1.2. Mucin type classification of O-linked glycopeptides

[0119] The polypeptides contained in the sample prepared in Example 1.1 were connected to a high-resolution mass spectrometer, Orbitrap Fusion lumos (Orbitrap Fusion™), and LC / ESI-MS / MS analysis was performed. To verify the reproducibility of the results, LC / MS / MS was performed three times in the following manner. Specifically, a C18 (3 μm, 75 μm x 2 cm) trap column and a C18 (2 μm, 75 μm x 50 cm) analysis column were used. The sample passing through the column was ionized by the principle of electrospray ionization (ESI), and the mass of the ions was measured using an Orbitrap Fusion Lumos mass spectrometer (Thermo). The analysis was performed at 40°C and a flow rate of 250 nℓ / min by injecting 5 μl of sample. Mobile phase A used in the above analysis was an aqueous solution containing 0.1% (v / v) formic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) formic acid. The analysis was performed three times by repeating LC / MS / MS under mobile phase concentration gradient conditions for 180 minutes.

[0120] The mass spectrometry result files (RAW) were converted to ms1 (MS) and ms2 (MS / MS) files using the freeware program RAWConverter v1.1 (The Scripps Research Institute, USA). The O-linked glycopeptide type identification method for mucin type classification was used to classify the types of O-linked glycopeptide candidates in each mass spectrometry result.

[0121] Example 1.3. Classification of glycopeptides according to GGRatio

[0122] The LC-MS / MS analysis results obtained above were used to derive the peak and intensity values ​​of oxonium ions, B ions, and Y ions. Based on these, the GGRatio value was derived according to the mathematical equation 2 described above. As a result, it was confirmed that glycated peptides were distinguished according to the GGRatio value (Figs. 5a and 5b).

[0123] Example 2. Analysis of hemopexin standard samples

[0124] Example 2.1. Preparation of hemopexin standard sample

[0125] Hemopexin standard samples were purchased from Sigma Alderrich. The purchased hemopexin sample was suspended in 100 μl of 50 mM ammonium bicarbonate. Trypsin was added in the same manner as in Example 1 above, and the sample was hydrolyzed overnight at 37°C. The hydrolyzed sample was concentrated using a ZIC®-HILIC column and prepared. The sample was processed in the same manner as in Example 1.2 above, and LC-MS / MS analysis was repeated three times. The results obtained by performing LC / MS / MS mass spectrometry three times were used to perform a qualitative analysis of O-linked glycopeptides using a method for analyzing the mucin type and sugar chain structure of O-linked glycopeptides.

[0126] Example 2.2. Analysis of hemopexin standard sample

[0127] The results of representative mucin types and sugar chain structures among the O-linked glycopeptides identified in Example 2.1 are shown in Figs. 6a and 6b. The O-linked glycopeptides are indicated by the number of representative monosaccharides constituting the O-glycan chain, in the order of Hexose, HexNAc, Fucose, and NeuAc. For example, peptide_2_2_1_2 refers to a glycopeptide composed of 2 Hexoses, 2 HexNAc, 1 Fucose, and 2 NeuAc. The O-linked glycopeptide identified in Fig. 6a was a glycopeptide identified with the configuration 2_2_0_3 in "TPLPPTSAHGNVAEGETKPDPDVTER (SEQ ID NO: 1)". However, as a result of analysis using the above O-linked glycopeptide analysis method, the glycopeptide identified in Fig. 6a was identified as an O-linked glycopeptide composed of the mucin type Core 1 type with 1_1_0_1 at the first amino acid “T” and 1_1_0_2 at the seventh amino acid “S.”

[0128] And, in Fig. 6b, the O-linked glycopeptide was identified as a glycopeptide composed of 2_2_1_2 in "TPLPPTSAHGNVAEGETKPDPDVTER". However, as a result of analysis using the above O-linked glycopeptide analysis method, the glycopeptide identified in Fig. 6b was identified as an O-linked glycopeptide composed of the mucin type Core 2 type with 2_2_1_2 in the first amino acid "T" in the peptide sequence.

Claims

1. 1) Step of acquiring mass spectrum data for a sample; 2) A step of selecting peptide spectrum data using tandem spectrum (MS / MS) data extracted from the above mass spectrum data; 3) A step of selecting O-linked glycopeptides from the selected glycopeptide spectrum data; 4) A step of obtaining information on GGRatio, B ion peak and Y ion peak from the selected peptide spectrum data; 5) A method for classifying O-linked glycopeptide types, comprising the step of classifying sample peptides into eight types according to the following conditions: At this time, if GGRatio<1.81, it is judged to be an O-linked glycopeptide of Core 1 type, If 1.81≤GGRatio<4 and there is no B ion peak for 2HexNAc (m / z 407.15) and no Y ion peak for PEP+2HexNAc, it is judged to be an O-linked glycopeptide of the Extended Core 1 type. If 1.81≤GGRatio<4.37 and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc, and there is no Y ion peak for PEP+HexHexNAc, it is judged to be an O-linked glycopeptide of the Core 2 type. If GGRatio≥4.37 and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc and a Y ion peak for PEP+HexHexNAc, it is judged to be an O-linked glycopeptide of the Extended Core 2 type. If GGRatio<4.37 and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc, there is no Y ion peak for PEP+HexHexNAc, and there is no B ion peak for 3HexNAc (m / z 610.72), it is judged to be an O-linked glycopeptide of the Core 3 type. If GGRatio≥4.37, and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc, there is no Y ion peak for PEP+HexHexNAc, and there is no B ion peak for 3HexNAc (m / z 610.72), it is judged to be an O-linked glycopeptide of the Extended Core 3 type. If GGRatio<4.37, and there is a B ion peak for 2HexNAc (m / z 407.15) or a Y ion peak for PEP+2HexNAc, there is no Y ion peak for PEP+HexHexNAc, and there is a B ion peak for 3HexNAc (m / z 610.72), it is judged to be an O-linked glycopeptide of the Core 4 type. At this time, if GGRatio≥4.37, a B ion peak for 2HexNAc or a Y ion peak for PEP+2HexNAc exists, a Y ion peak for PEP+HexHexNAc does not exist, and a B ion peak for 3HexNAc (m / z 610.72) exists, it is judged to be an O-linked glycopeptide of the Extended Core 4 type.

2. In paragraph 1, The above GGRatio is an O-linked glycopeptide type classification method obtained by the following [Mathematical Formula 2]: [Equation 2] Here, And, 138, 168, 126 and 144 represent the mass-to-charge ratio (m / z) for specific peaks, Ii is the intensity of the observed ith peak, Imax is base peak intensity (BPI).

3. In paragraph 1, O-linked glycopeptide type classification method, wherein the O-linked glycopeptide type has the following GlcNAc number and HexNAc number: O-linked glycopeptides of Core 1 type have GlcNAc=0 ​​and HexNAc=1, O-linked glycopeptides of the Extended Core 1 type are GlcNAc≥1 and HexNAc=2, O-linked glycopeptides of the Core 2 type are GlcNAc=1 and HexNAc=2. O-linked glycopeptides of the Extended Core 2 type are GlcNAc≥2 and HexNAc=3, Core 3 type O-linked glycopeptides are GlcNAc=1 and HexNAc=2. O-linked glycopeptides of the Extended Core 3 type are GlcNAc≥2 and HexNAc=3, Core 4 type O-linked glycopeptides are GlcNAc=2, HexNAc=3, and O-linked glycopeptides of the Extended Core 4 type are GlcNAc≥3 and HexNAc=4.

4. In paragraph 1, The selection of the peptide spectrum data in the above step 2) is performed by calculating the M-score from the tandem spectrum (MS / MS) data according to the following [Mathematical Formula 1], a method for classifying O-linked peptide types: [Mathematical Formula 1] Here, And, N is the total number of multiple oxonium ion peaks, n is the number of oxonium ions identified in the spectral data of the sample, Ii is the intensity of the observed ith peak, Imax is the base peak intensity (BPI), C is a constant value.

5. In paragraph 4, A method for classifying O-linked glycopeptide types, wherein the above plurality of oxonium ion peaks correspond to Table 1.

6. In paragraph 1, A method for classifying O-linked glycopeptide types, wherein the sample is hydrolyzed with any one enzyme selected from the group consisting of trypsin, chymotrypsin, and proteinase K.

7. In paragraph 1, A method for classifying O-linked glycopeptide types, wherein the above mass spectrum data is obtained by a mass spectrometer having a mass resolution of 10,000 or more and a mass accuracy of 50 ppm or less. 8.1) When the condition dHN=0 is satisfied, a step of determining that the glycosylation site is one; and 2) A method for analyzing the number of sugar chains in an O-linked glycopeptide, including a step of determining that there are multiple glycosylation sites when the condition pNGS≤NGS is satisfied; The above dHN is the HexNAc number derived according to [Mathematical Formula 3] below, [Equation 3] dHN = nHN - pHN The above nHN is the number of HexNAc of O-glycopeptides observed in the sample; The above pHN represents the number of HexNAc predicted according to the following conditions (i) to (iv): (i) If GGRatio≤1.8, HexNAc is 1, (ii) 1.81 <GGRatio≤4.37 인 경우, HexNAc는 2이며, (iii) 4.37 <GGRatio≤7.0 인 경우, HexNAc는 3이며, 및 (iv) 7.0 <GGRatio인 경우, HexNAc는 4이며; The above pNGS is the number of observable O-glycosylation sites derived according to the following [Mathematical Formula 4], [Equation 4] pNGS = dHN + 1, The above NGS indicates the number of O-glycosylation sites in the non-glycosylated sample.

9. In paragraph 8, A method for analyzing the number of sugar chains in an O-linked glycopeptide, wherein the number of Thr and Ser in the above-mentioned non-glycosylated sample is determined to be capable of having an O-glycosylation site.

10. In paragraph 9, A method for analyzing the number of sugar chains in an O-linked glycopeptide, wherein the non-glycosylated sample may have five O-glycosylation sites when TPLPPTSAHGNVAEGETKPDPDVTER (SEQ ID NO: 1).

11. A step of deriving B ion, Y ion and O-linked glycopeptide types from the first paragraph; A step of deriving the number of sugar chains in an O-linked glycopeptide from the 8th clause; and A method for analyzing O-linked glycopeptides, comprising a step of analyzing O-linked glycopeptides of [Table 2] based on O-linked glycopeptide type, B ion, and Y ion peak values.

12. A program applied to perform a method for classifying O-linked glycopeptide types according to any one of claims 1 to 7; A program applicable to performing the method for analyzing the number of sugar chains in an O-linked glycopeptide of claim 8 or 9; and / or A computer-readable recording medium recording a program applicable to performing the O-linked glycopeptide analysis method of Article 11.

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