AU2014101378A4 - Glycomic biomarker of rat serum based on microfluidic chip-lc/ms - Google Patents

Abstract

The present invention relates to a method of characterizing the serum N-glycome using microfluidic chip-LC-ESI-QTOF MS and MS/MS techniques for selection of the most appropriate animal model for pathological and pharmacological studies. The N-glycans of rat serum were comprehensively profiled using the newly developed technique. In total, 282 N glycans, included isomers were identified among which 172 glycans were reported from rat serum for the first time, and 27 were novel glycans. Cl C) o U z o z z C.) U z o '0 o 0 - _____ oN Cl -z ~ rfl (~) -+ - - _________________ ____ ____ -2 ~flN Cl _ .4 -~ -~ C-) '0 z z Cl '0 t N ~r q~ - q~ tin C ~ 'p t r - CC '0 t Cl C o C-r ~t ~r1 o - - - C C 8 8 (s~uno~ uci) Avsueiui (siunco uoi) 4suemi o a Cl 7 U e 0 U ~: -o C-) U r. z z x '0 '0 o ooooooo66 C 666o (s~unoj uoi) k!sueluI (siunos uoi) A1!sue~uI

Description

GLYCOMIC BIOMARKER OF RAT SERUM BASED ON MICROFLUIDIC CHIP LC/MS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from the US provisional patent application serial number 62/064,487 filed October 16 th, 2014, and the disclosure of which is incorporated herein by reference in its entirety. FIELD OF INVENTION The present invention relates to a method of characterizing the serum N-glycome using microfluidic chip-LC-ESI-QTOF MS and MS/MS techniques for selection of the most appropriate animal model for pathological and pharmacological studies. BACKGROUND OF INVENTION Owing to the inherent similarity to humans, rats have been used as subjects for scientific research in many experimental studies that have contributed significantly to our understanding of a variety of scientific areas including genetics, human diseases, and pharmacological studies. Among rodents, rats are physiologically most similar to humans. For instance, previous studies such as Abbott A (2004) Laboratory animals: the Renaissance rat. Nature, 428(6982): 464-466, shows the heart rate of a rat is similar to that of a human, whereas the heart rate of a mouse is about five to ten times faster. Additionally, the detoxifying enzymes of a rat are highly similar to those of a human. A rat model is therefore superior to a mouse model for assaying the pharmacodynamics and toxicities of drugs and for studying various human diseases, such as cardiovascular disease, arthritis, diabetes mellitus, and autoimmune disorders, and numerous rat models have been established for such types of in vivo research. The N-glycans of glycoproteins are directly involved in almost every biological process and play a crucial role in human diseases. Glycomic studies therefore substantially impact on research towards diagnosing, preventing, and treating diseases [3-6]. For example, glycomic studies of liver cancer in rats revealed that variations in fucosylation of glycans in serum glycoproteins were closely related to the progression of hepatocellular carcinomas. This was consistent with observations for human liver cancer [7-9]. However, in comparison to the 1 vast array of research into the human glycome, limited studies have been performed on the rat glycome. Less than 50 N-glycan compositions of rat serum have been reported to date [7, 8]. Therefore, a comprehensive glycomic study in rats is much needed. The findings from which may aid our understanding of the physiology and pathology associated with glycosylation for various diseases. SUMMARY OF INVENTION The present invention features a method for characterizing the serum N-glycome using microfluidic chip-LC-ESI-QTOF MS and MS/MS techniques for selection of the most appropriate animal model for pathological and pharmacological studies. The main aspect of the present invention provides a method for detecting and identifying glycomic biomarkers, especially unknown biomarkers and those in low abundance, from serum of a mammal for selection of most appropriate animal model for pathological and pharmacological studies for a subject. The method of detecting and identifying mainly comprises sample preparation, liquid chromatography - mass spectrometry, and comparison of glycomic profiles from serum between the mammal and the subject. In the presently claimed method, the sample preparation step further comprises thermal denaturation in SDS-containing buffer with addition of DTT; digestion by N-glycosidase F (PNGase F); and purification by Hypercarb solid-phase extraction (SPE) cartridges. In the presently claimed method, the liquid chromatography - mass spectrometry step further comprises a microfluidic porous graphitized carbon (PGC) chip-LC/MS with Mobile phase condition of 0.5% formic acid of pH 3.0 in water and acetonitrile. In the preferred embodiment, the glycomic biomarkers are N-glycans. In the presently claimed method, the comparison step of serum N-glycans are divided into five classes comprising (1) high mannose N-glycans, (2) undecorated complex/hybrid N glycans, (3) fucosylated complex/hybrid N-glycans, (4) sialylated complex/hybrid N-glycans, and (5) fucosylated-sialylated complex/hybrid N-glycans. 2 In a preferred embodiment, the mammal from which serum is compared comprises rat serum when the subject is either human or mouse. In another embodiment, the rat serum which comprises 129 N-acetylneuramic acid and 59 N glycolylneuramic acid is more similar to human serum than mouse serum based on high abundance of N-acetylneuramic acid and similar glycomic distributions. In other embodiment, 282 N-glycans are identified in the rat serum, which comprises 192 acidic glycans and 90 neutral glycan. In a further embodiment, the 192 acidic glycans so identified further comprises 129 N acetylneuraminic acid, 59 N-glycolylneuraminic acid and 4 N-glycans wherein N acetylneuraminic acid and N-glycolylneuraminic acid co-exist. The 129 N-acetylneuramic acid further comprises 102 N-glycans containing 0-accetylated sialic acid and 27 novel glycans. In yet another embodiment, the degree of sialylation in the N-glycans varies from one to four with di-sialylated N-glycans as the most dominant species; the degree of O-acetylation in the N-glycans at distinct sites varies from one to three with mono-acetylation being the most dominant species, and the 0-accetylated N-glycans may be involved in a variety of biological processes in mammals including lectin recognition, virus binding, tumor antigenicity, tissue morphogenesis, and cell-cell interactions, which are associated with various human diseases. Those skilled in the art will appreciate that the present invention described herein is susceptible to variations and modifications other than those specifically described. The present invention includes all such variation and modifications. The present invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of 3 integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of' and "consists essentially of' can allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the present invention. Furthermore, throughout the specification and claims, unless the context requires otherwise, the word "include" or variations such as "includes" or "including", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Other definitions for selected terms used herein may be found within the detailed description of the present invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present invention belongs. Other aspects and advantages of the present invention will be apparent to those skilled in the art from a review of the ensuing description. BRIEF DESCRIPTION OF FIGURES FIG. 1 Differentiation of isobaric glycans based on the accurate mass value. FIG. 2 overlaid extracted compound chromatograms (ECC) of rat serum N-glycans identified by PGC nano-LC/MS FIG. 3 Targeted MS/MS of sialylated N-glycans in rat serum. (A) Characteristic fragment ions derived from N-glycans containing NeuAc (Left), NeuGc (Middle), and both NeuAc and NeuGc (Right). (B) Full MS/MS spectra of rat serum N-glycan Hex 5 HexNAc 4 NeuAc 1 NeuGci (doubly protonated m/z 1120.47). FIG. 4 Targeted MS/MS of O-acetylated N-glycans in rat serum. (A) Diagnostic fragment ions generated from N-glycans with O-acetylated NeuAc (Left) and O-acetylated NeuGc (Right). (B-E) Full MS/MS spectra of a series of N-glycans with varied numbers (0-3) of acetylation sites on NeuAc. FIG. 5 MS/MS spectra of rat serum N-glycan Hex 5 HexNAc 4 NeuAc 1 NeuGc 1 +OAc (doubly protonated m/z 1141.40) (A) and proposed fragmentation (B). 4 FIG. 6 The overall relative abundances of 97 distinct N-glycan compositions in rat, mouse, and human sera (A) and relative abundances of each N-glycan class (B). High mannose (HM), undecorated complex/hybrid (C/H), fucosylated complex/hybrid (C/H-Fuc), sialylated complex/hybrid (C/H-Sia), and fucosylated/sialylated complex/hybrid (C/H-Fuc&Sia). For the full name of each N-glycan refer to Table 1. FIG. 7 Species-specific variations of N-glycans with different numbers of sialic acids (A), with or without fucose (B), and with different numbers of antennary (C), in rat, mouse, and human sera. FIG. 8 Variant relative abundance of N-glycans with NeuAc or NeuGc, or with both NeuAc and NeuGc in rat, mouse, and human sera. FIG. 9 The relative abundance of 0-acetylated N-glycans in rat serum (A) and the retention behavior of 0-acetylated N-glycans on the PGC chip (B). DETAILED DESCRIPTION OF INVENTION The rat is an important alternative for studying human pathology owing to certain similarities to humans. Glycomic studies on rat serum have revealed that variations in the glycans of glycoproteins correlated with disease progression, which is consistent with the findings in human serum. However, few reports have analyzed N-glycans in rat serum. The present invention relates to identifying and characterizing rat serum N-glycome for the first time using microfluidic chip-LC-ESI-QTOF MS and M/MS techniques for selection of the most appropriate animal model for pathological and pharmacological studies. The present invention is further illustrated by the following examples, which should not be construed as further limiting. Example 1 - Collection of serum samples Sera of male Sprague-Dawley rats weighing 200-250 g and male mice weighing 20-22 g were collected via orbital eye bleeding. Sera from volunteers were obtained from the Guangdong Provincial Chinese Medicine Hospital. This study was approved by the Ethics Committee of Guangdong Provincial Chinese Medicine Hospital. Example 2 - Preparation of N-glycan sample for glycomic profiling The initial step for glycan analysis is the release of glycans from glycoproteins. N-glycan release and purification were performed according to the PNGase F protocol provided by 5 New England Biolabs (Ipswich, MA, USA). Briefly, 5 [tL of serum were thermally denatured in denaturing buffer containing 0.5% SDS and 40 mM DTT, 10 min prior to digestion by PNGase F (New England Biolabs). Digestion was performed in 50 mM sodium phosphate and 1% NP-40 overnight at 37'C. Released N-glycans were purified by Hypercarb solid phase extraction (SPE) cartridges, washed with water, then eluted with 40% acetonitrile and 0.05% trifluoroacetic acid (v/v) in water. Example 3 - Separation of N-glycans on the microfluidc PGC chip Samples were analyzed using a microfluidic chip-LC coupled with the Agilent 6550 iFunnel Accurate Mass Quadrupole Time-of-Flight Mass Spectrometer System (Agilent Technologies, Santa Clara, CA, USA) equipped with an auto-sampler (maintained at 5'C), capillary pumps, nano pumps, and a chip/MS interface. The microfluidic chip consisted of a 40-nL enrichment column and a 43xO.075 mm i.d. analytical column, both packed with 5 [tm graphitized carbon as the stationary phase, with integrated nano-ESI spray tips. For each sample, 1.0 [tL of sample solution was loaded onto the enrichment column and washed with a solution of 0.1% formic acid (v/v) in water. A rapid N-glycan elution gradient was delivered at 0.5 [tL/min using solutions of (A) 65 mM formic acid buffered to pH 3 in water and (B) 100% acetonitrile, at the following proportions and time points: 5-60%, 0-12 min; Remaining non glycan compounds were flushed out with 80% B at 0.5 [tL/min for 3 min, while the enrichment column was re-equilibrated with 0.1% formic acid at 3 [tL/min for 10 min. The drying gas temperature was set at 225'C with a flow rate of 11 L/min (filtered nitrogen gas). MS spectra were acquired over a mass range of m/z 500-3000 with an acquisition time of 1.0 s per spectrum in positive ionization mode. The instrument was operated using the target MS/MS mode, with the m/z range from m/z 50 to 3000 with an acquisition time of 1.5 s per spectrum. Mass correction was enabled using a reference mass of m/z 922.0098 as the internal standard (G1969-85001; Agilent Technologies). The collision energy was set at 5-20 V. The full width half maximum of the quadrupole mass bandpass used during MS/MS precursor isolation was set to medium (-4 m/z). Example 4 - characterization of N-glycans based on accurate mass LC/MS raw data were processed using the Molecular Feature Extractor (MFE) algorithm (Version B.06.00; Agilent Technologies). MFE is able to perform chemical relationship 6 testing and chromatographic covariance testing, identify charge carriers (such as sodium) and multimers and group them, and reconstruct spectra by including isotope information. The critical parameters setting in the MFE includes the N-glycan model in the isotope model item, three as the maximum charge states, and 20 ppm as the accurate mass criteria. MS peaks were filtered with a signal-to-noise ratio of 5.0 and parsed into individual ion species. Using the expected isotopic distribution, charge state information and retention time, all ion species associated with a single compound (e.g., the doubly protonated ion, the triply protonated ion, and all associated isotopologues) were totaled, and the neutral monoisotopic mass of the compound was calculated. Using this information, a list of all peaks in the sample was generated with abundances represented by chromatographic peak areas. Computerized algorithms were used to identify N-glycan compositions by accurate mass. By combining these empirical findings with previous research into the N-glycans of mammals, a virtual personal compound database and library (PCDL) was established resulting in over 4000 N glycan compositions, which contained all biologically plausible rat serum N-glycan compositions with modifications such as O-acetylation, methylation, lactylation, sulfation, phosphorylation, and glycosylation. Deconvoluted experimental masses were compared against theoretical N-glycan masses using a mass error tolerance of 5 ppm. The scoring of the generated formulas was based on three factors: first, the measured mass (or m z) was compared with the value predicted from the proposed formula; second, the abundance pattern of the measured isotope cluster was compared with values predicted from the proposed formula; third, the m z spacing between the lowest m z ion and the A+1 and A+2 ions were compared with the values predicted from the proposed formula. These individual factors were computed as match probabilities. Combining the individual match probabilities into an overall score was done as a weighted average rather than as a product. On the basis of known rat serum N-glycosylation patterns, N-glycan compositions containing hexose (Hex), N acetylhexosamine (HexNAc), deoxyhexose (dHex), N-acetylneuraminic acid (NeuAc), N glycolylneuraminic acid (NeuGc), and O-acetylation (OAc) were considered. The ultra-high accurate mass value obtained by Q-TOF MS facilitated assignment of N glycans at the MS level. In particular, isobaric glycans can be well differentiated based on the high-resolution MS data. For example, the N-glycan at m z 1128.3934 was identified as disialylated N-glycan Hex 5 HexNAc 4 NeuGc 2 based on its mass value [M+2H] 2 +

(C

8 4 H13 8

N

6

O

6 4 , cal. m/z 1128.3937), while the N-glycan atm z 1128.9020 was characterized as Hex 6 HexNAc 4 dHex 1 NeuGci on the basis of its mass value [M+2H] 2 + (C 8 5

H

14 1

N

5 0 6 4 , 7 theoretical m/z 1128.9039). Both N-glycans could be unambiguously distinguished according to their accurate mass values (FIG. 1). In total, 282 glycans including 192 acidic glycans and 90 neutral glycans were identified among which 172 glycans were reported from rat serum for the first time, and 27 were novel glycans (FIG. 2). Table 1 show the N-glycans identified in rat, mouse and human sera. Table 1: Accurate Full name Abbreviation Formula Molecular Weight Hex 1 HexNAc 2 1_2_0_0 C22 H38 N2 016 586.2050 Hex 2 HexNAc 2 2_2_0_0 C28 H48 N2 021 748.2702 Hex 3 HexNAc 2 3_2_0_0 C34 H58 N2 026 910.3249 Hex 4 HexNAc 2 4_2_0_0 C40 H68 N2 031 1072.3531 Hex 3 HexNAc 3 3_3_0_0 C42 H71 N3 031 1113.4032 Hex 5 HexNAc 2 5_2_0_0 C46 H78 N2 036 1234.4313 Hex 3 HexNAc 3 dHex 1 3_3_1_0 C48 H81 N3 035 1259.4618 Hex 4 HexNAc 3 4_3_0_0 C48 H81 N3 036 1275.4561 Hex 3 HexNAc 4 3_4_0_0 C50 H84 N4 036 1316.4806 Hex 6 HexNAc 2 6_2_0_0 C52 H88 N2 041 1396.4860 Hex 4 HexNAc 3 dHex 1 4_3_1_0 C54 H91 N3 040 1421.5158 Hex 5 HexNAc 3 5_3_0_0 C54 H91 N3 041 1437.5128 Hex 3 HexNAc 4 dHex 1 3_4_1_0 C56 H94 N4 040 1462.5436 Hex 4 HexNAc 4 4_4_0_0 C56 H94 N4 041 1478.5380 Hex 3 HexNAC 5 3_5_0_0 C58 H97 N5 041 1519.5630 Hex 7 HexNAc 2 7_2_0_0 C58 H98 N2 046 1558.5353 Hex 4 HexNAc 3 NeuAci 4_3_0_1* C59 H98 N4 044 1566.5529 Hex 4 HexNAc 3 NeuGci 4_3_0_1# C59 H98 N4 045 1582.5545 Hex 5 HexNAc 3 dHex 1 5_3_1_0 C60 H101 N3 045 1583.5707 Hex 6 HexNAc 3 6_3_0_0 C60 H101 N3 046 1599.5656 Hex 4 HexNAc 3 NeuAc 1 +OAc 4_3_0_1*Xa C61 H 100 N4 045 1608.5653 Hex 4 HexNAc 4 dHex 1 4_4_1_0 C62 H104 N4 045 1624.5973 Hex 5 HexNAc 4 5_4_0_0 C62 H104 N4 046 1640.5896 Hex 4 HexNAc 3 NeuAc 1 +2OAc 4_3_0_1* Sb C63 H 102 N4 046 1650.5765 8 Hex 3 HexNAC 5 dHex 1 3_5_1_0 C64 H 107 N5 045 1665.6231 Hex 4 HexNAC 5 4_5_0_0 C64 H 107 N5 046 1681.6142 Hex 4 HexNAc 3 dHex 1 NeuAci 4_3_1_1* C65 H108 N4 048 1712.6091 Hex 8 HexNAc 2 8_2_0_0 C64 H108 N2 051 1720.5833 Hex 4 HexNAc 3 dHex 1 NeuGci 4_3_1_1# C65 H108 N4 049 1728.6061 Hex 6 HexNAc 3 dHex 1 6_3_1_0 C66 H 111 N3 050 1745.6235 Hex 7 HexNAc 3 7_3_0_0 C66 H 111 N3 051 1761.6184 Hex 4 HexNAc 4 NeuAci 4_4_0_1* C67 H 111 N5 049 1769.6359 Hex 4 HexNAc 4 NeuGci 4_4_0_1# C67 H 111 N5 050 1785.6183 Hex 5 HexNAc 4 dHex 1 5_4_1_0 C68 H 114 N4 050 1786.6498 Hex 6 HexNAc 4 6_4_0_0 C68 H 114 N4 051 1802.6456 Hex 4 HexNAc 4 NeuAc 1 +0Ac 4_4_0_1*Xa C69 H 113 N5 050 1811.6393 Hex 4 HexNAC 5 dHex 1 4_5_1_0 C70 H117 N5 050 1827.6763 Hex 5 HexNAC 5 5_5_0_0 C70 H117 N5 051 1843.6655 Hex 9 HexNAc 2 9_2_0_0 C70 H118 N2 056 1882.6447 Hex 6 HexNAc 3 NeuAci 6_3_0_1* C71 H118 N4 054 1890.6610 Hex 4 HexNAc 4 dHex 1 NeuAci 4_4_1_1* C73 H121 N5 053 1915.6917 Hex 5 HexNAc 4 NeuAci 5_4_0_1* C73 H121 N5 054 1931.6883 Hex 5 HexNAc 4 NeuGci 5_4_0_1# C73 H121 N5 055 1947.6813 Hex 6 HexNAc 4 dHex 1 6_4_1_0 C74 H124 N4 055 1948.7079 Hex 7 HexNAc 4 7_4_0_0 C74 H124 N4 056 1964.6987 Hex 4 HexNAC 5 NeuAci 4_5_0_1* C75 H124 N6 054 1972.7131 Hex 5 HexNAc 4 NeuAc 1 +OAc 5_4_0 1*Xa C75 H123 N5 055 1973.6922 Hex 5 HexNAC 5 dHex 1 5_5_1_0 C76 H127 N5 055 1989.7281 Hex 6 HexNAC 5 6_5_0_0 C76 H 127 N5 056 2005.7244 HexioHexNAc 2 10_2_0_0 C76 H128 N2 061 2044.6975 Hex 5 HexNAc 4 dHex 1 NeuAci 5_4_1_1* C79 H131 N5 058 2077.7464 Hex 5 HexNAc 4 dHex 1 NeuGci 5_4_1_1# C79 H131 N5 059 2093.7381 Hex 7 HexNAc 4 dHex 1 7_4_1_0 C80 H134 N4 060 2110.7557 Hex 4 HexNAC 5 dHex 1 NeuAci 4_5_1_1* C81 H134N6 058 2118.7697 Hex 5 HexNAc 4 dHex 1 NeuAc 1 +OAc 5_4_1 _1*Xa C81 H133 N5 059 2119.7510 Hex 4 HexNAC 5 dHex 1 NeuGci 4_5_1_1# C81 H134 N6 059 2134.7670 Hex 5 HexNAC 5 NeuAci 5_5_0_1* C81 H134 N6 059 2134.7675 Hex 5 HexNAc 4 dHex 1 NeuGc 1 +OAc 5_4_1_1#Xa C81 H133 N5 060 2135.7600 9 Hex 6 HexNAC 5 dHex 1 6_5_1_0 C82 H137 N5 060 2151.7731 Hex 5 HexNAc 4 dHex 1 NeuAc 1 +2OAc 5_4_1_1*Xa C83 H135 N5 060 2161.7651 Hex 5 HexNAc 4 NeuAc 2 5_4_0_2* C84 H138 N6 062 2222.7835 Hex 5 HexNAc 4 NeuAc 1 NeuGci 5_4_0_2*# C84 H138 N6 063 2238.7793 Hex 5 HexNAc 4 NeuGc 2 5_4_0_2# C84 H138 N6 064 2254.7718 Hex 5 HexNAc 4 NeuAc 2 +0Ac 5_4_0_2* Xa C86 H140 N6 063 2264.7937 Hex 5 HexNAc 4 NeuAc 1 NeuGc 1 +0Ac 5_4_0_2*#a C86 H140 N6 064 2280.7885 Hex 5 HexNAC 5 dHex 1 NeuAci 5_5_1_1* C87 H144 N6 063 2280.8247 Hex 6 HexNAC 5 NeuAci 6_5_0_1* C87 H144 N6 064 2296.8156 Hex 5 HexNAc 4 NeuAc 2 +20Ac 5_4_0_2* Xb C88 H142 N6 064 2306.8048 Hex 5 HexNAc 4 NeuAc 2 +30Ac 5_4_0_2*Xc C90 H144 N6 065 2348.8147 Hex 5 HexNAc 4 dHex 1 NeuAc 2 5_4_1_2* C90 H148 N6 066 2368.8402 Hex 5 HexNAc 4 dHex 1 NeuAc 1 NeuGci 5_4_1_2*# C90 H148 N6 067 2384.8354 Hex 5 HexNAc 4 dHex 1 NeuGc 2 5_4_1_2# C90 H148 N6 068 2400.8290 Hex 5 HexNAc 4 dHex 1 NeuAc 2 +OAc 5_4_1_2* Xa C92 H150 N6 067 2410.8465 Hex 5 HexNAC 5 NeuAc 2 5_5_0_2* C92 H151 N7 067 2425.8539 Hex 5 HexNAc 4 dHex 1 NeuAc 1 NeuGc 1 +OAc 5_4_1_2*#a C92 H150 N6 068 2426.8495 Hex 5 HexNAC 5 NeuAciNeuGci 5_5_0_2*# C92 H151 N7 068 2441.8539 Hex 6 HexNAC 5 dHex 1 NeuAci 6_5_1_1* C93 H154 N6 068 2442.8736 Hex 5 HexNAc 4 dHex 1 NeuAc 2 +2OAc 5_4_1_2* Xb C94 H152 N6 068 2452.8623 Hex 5 HexNAc 4 dHex 1 NeuAc 2 +3OAc 5_4_1_2* Xc C96 H154 N6 069 2494.8623 Hex 5 HexNAc 4 NeuAc 3 +OAc 5_4_0_3* Xa C97 H157 N7 071 2555.8890 Hex 5 HexNAC 5 dHex 1 NeuAc 2 5_5_1_2* C98 H161 N7 071 2571.9193 Hex 6 HexNAC 5 NeuAc 2 6_5_0_2* C98 H161 N7 072 2587.9117 Hex 5 HexNAc 4 NeuAc 3 +2OAc 5_4_0_3* Xb C99 H159 N7 072 2597.8996 Hex 6 HexNAC 5 NeuAciNeuGci 6_5_0_2*# C98 H161 N7 073 2603.9101 Hex 6 HexNAC 5 NeuGc 2 6_5_0_2# C98 H161 N7 074 2619.9050 Hex 5 HexNAc 4 NeuAc 3 +3OAc 5_4_0_3*Xc C1O H161 N7 073 2639.9101 Hex 5 HexNAc 4 dHex 1 NeuAc 3 +OAc 5_4_1_3*-a C103 H 167 N7 075 2701.9469 Hex 6 HexNAC 5 dHex 1 NeuAc 2 6_5_1_2* C104 H 171 N7 076 2733.9620 Hex 5 HexNAc 4 dHex 1 NeuAc 3 +2OAc 5_4_1_3*-b C105 H 169 N7 076 2743.9575 Hex 5 HexNAc 4 dHex 1 NeuAc 3 +3OAc 5_4_1_3*Xc C107 H 171 N7 077 2785.9680 Hex 6 HexNAC 5 NeuAc 3 6_5_1_3* C109 H178 N8 080 2879.0106 Hex 6 HexNAC 5 NeuAc 3 +OAc 6_5_0_3*a C1II H180N8 081 2921.0212 10 Hex 7 HexNAc 6 NeuAc 2 7_6_0_2* C112 H184N8 082 2953.0474 Hex 6 HexNAC 5 NeuAc 3 +2OAc 6_5_0_3*Xb C113 H182 N8 082 2963.0318 Hex 6 HexNAC 5 NeuAc 3 +3OAc 6_5_0 3*Xc C115 H184 N8 083 3005.0423 Hex 7 HexNAC 6 dHex 1 NeuAc 2 7_6_1_2* C118 H194 N8 086 3099.1053 Hex 7 HexNAc 6 NeuAc 3 7_6_0_3* C123 H201 N9 090 3244.1428 Hex 6 HexNAC 5 NeuAc 4 +20Ac 6_5_0_4* b C124 H 199 N9 090 3254.1272 Hex 6 HexNAC 5 NeuAc 4 +30Ac 6_5_0_4*Xc C126 H201 N9 091 3296.1378 Hex 7 HexNAC 6 dHex 1 NeuAc 3 7_6_1_3* C129 H211 N9 094 3390.2007 Hex 7 HexNAc 6 NeuAc 4 7_6_0_4* C134 H218 N10 098 3535.2353 Hex 7 HexNAc 6 dHex 1 NeuAc 4 7_6_1_4* 0140 H228 N10 3681.2961 * NeuAc; # NeuGc; X O-acetylation, a mono-, b di-, c tr Example 5 - MS/MS screening for NeuAc- and NeuGc- containing N-glycans N-glycolylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) represent two of the most common sialic acids in nature. N-glycans containing NeuAc and NeuGc were widespread on all mammalian cell surfaces in a species-specific manner, and were associated with pathological conditions. This indicates the importance of identification of NeuAc and NeuGc. The only difference between the two sialic acids, NeuAc and NeuGc, is an additional oxygen atom in the N-glycolyl group of NeuGc (acetyl amino group). Targeted MS/MS experiments are carried for differentiating these types of isomers. Fragment ions derived from respective sialic acids, i.e., product ions at m z 274.09 (NeuAc-H 2 0), m z 292.10 (NeuAc), and m z 657.23 (HexiHexNAciNeuAci) generated from NeuAc glycan, as well as fragment ions at m z 290.08 (NeuGc-H 2 0), m z 308.09 (NeuGc), and m z 673.22 (HexiHexNAciNeuGci) derived from NeuGc glycan, were employed as diagnostic ions for differentiation of these two species (FIG. 3A). This approach facilitated the identification of a large number of sialylated N-glycans, among which 129 were NeuAc glycans, 59 were NeuGc glycans, and four were 'mixed' glycans containing both NeuAc and NeuGc. Among the four mixed N-glycans, i.e., Hex 5 HexNAc 4 NeuAc 1 NeuGci, Hex 5 HexNAc 4 NeuAc 1 NeuGc 1 +OAc, Hex 5 HexNAc 4 dHex 1 NeuAc 1 NeuGci, and Hex 5 HexNAc 4 dHex 1 NeuAc 1 NeuGc 1 +OAc (FIG. 3B). Example 6 - Screening for modified N-glycans 11 Additional modifications of N-glycans occur on the hydroxyl group of the monosaccharide, these may include O-acetylation, methylation, lactylation, sulfation, or phosphorylation. To survey the modifications of rat serum N-glycans, the aforementioned five types of modifications were all included in the PCDL for characterization. On the basis of accurate mass, isotopic abundance, and targeted MS/MS, 0-acetylation was found to be the dominant modification of rat serum N-glycans. Taking di-sialylated glycan Hex 5 HexNAc 4 NeuAc 2 and its mono-O-acetylation as an example, the identification procedures were performed as follows. First, N-glycans were identified based on PCDL. Because the accurate mass and isotopic abundance of compounds in the MS scan were highly similar to the theoretical data on PCDL, they were preliminarily identified as Hex 5 HexNAc 4 NeuAc 2 and Hex 5 HexNAc 4 NeuAc 2 +OAc. O-acetylation was verified by analysis of an associated fragment produced by targeted MS/MS. Regular variations were found by comparing the MS/MS spectrum of the two N-glycans (FIG. 4A). A series of fragments, such as m z 274.09, 292.10, and 657.23, shown in the Hex 5 HexNAc 4 NeuAc 2 MS/MS spectrum (FIG. 4B) indicated the existence of HexNAc+Hex+NeuAc, that is sequentially attached to the core structure of the N-glycan. For Hex 5 HexNAc 4 NeuAc 2 +OAc (FIG. 4C), the series of fragments were 42 Da more than the corresponding fragments in Hex 5 HexNAc 4 NeuAc 2 , i.e., m z 316.09 (274.09+42), 334.10 (292.10+42), and 699.24 (657.23+42). These characteristic ions facilitated identification of O-acetylated sialic acid in N-glycans. Using this approach, N-glycans Hex 5 HexNAc 4 NeuAc 2 with between one and three acetyl groups were identified (FIG. 4D and 4E). Uncommon disialylated N-glycans with three O-acetyl groups, Hex 5 HexNAc 4 NeuAc 2 +3OAc and Hex 5 HexNAc 4 dHex 1 NeuAc 2 +3OAc, were identified in rat serum using this approach. In these N-glycans, one O-acetylated group was located at one terminal NeuAc, while the other two 0-acetyl groups were located at another terminal NeuAc. This approach also led to the identification of a novel O-acetylated 'mixed' N-glycan possessing both NeuAc and NeuGc in the molecule, i.e., Hex 5 HexNAc 4 dHex 1 NeuAc 1 NeuGc 1 +OAc (m/z 1141.40) (FIG. 5). Acetylation-associated fragments are summarized in Table 2 and the characterized O-acetyl N-glycans of rat serum are listed in Table 3. Although both NeuAc and NeuGc exist in rat serum N-glycans, 0 acetylation exclusively occurs on NeuAc. 12 Table 2: No 0-acetylation Symbol Composition m/z charge * -H 2 0 NeuAc-H 2 0 274.09 1 4 NeuAc 292.10 1

-H

2 0 Hex 1 NeuAci-H 2 0 454.15 1 HexiHexNAciNeuAci 657.23 1 Hex 2 HexNAc 1 NeuAci 819.28 1 Hex 4 HexNAc 2 NeuAci 1346.47 1 Hex 3 HexNAc 3 NeuAci 1405.50 1 Hex 4 HexNAc 3 NeuAci 1567.56 1 Hex 5 HexNAc 4 NeuAci 966.85 2 Mono-O-acetylation Symbol Composition m/z Charge +OAc -H 2 0 NeuAc+OAc-H 2 0 316.09 1 0' +OAc NeuAc+OAc 334.10 1 +OAc-H 2 0 Hex 1 NeuAc 1 +OAc-H 2 0 496.17 1 +OAc-H 2 0 Hex 1 HexNAc 1 NeuAc 1 +OAc 699.24 1 +OAc-H 2 0 Hex 2 HexNAc 1 NeuAc 1 +OAc 861.30 1 +0Ac-H 2 0 Hex 4 HexNAc 2 NeuAc 1 +OAc 1388.48 1 Hex 3 HexNAc 3 NeuAc 1 +OAc 1447.52 1 +OAc-H 2 0 + +OAc- Hex 4 HexNAc 3 NeuAc 1 +OAc 1609.57 1 H20 Hex 5 HexNAc 4 NeuAc 1 +OAc 987.85 2 +OAc-H 2 0 13 Di-O-acetylation Symbol Composition m/z Charge 4 +20Ac -H 2 0 NeuAc+20Ac-H 2 0 358.11 1 +20Ac NeuAc+20Ac 376.12 1 +N +20Ac-H 2 0 Hex 1 NeuAc 1 +20Ac 538.18 1

-H

2 0 +20Ac-H 2 0 Hex 1 HexNAc 1 NeuAc 1 +20Ac 741.26 1 +20Ac-H 2 0 Hex 2 HexNAc 1 NeuAc 1 +2OAc 903.31 1 +20Ac-H 2 0 Hex 4 HexNAc 2 NeuAc 1 +20Ac 1430.49 1 Hex 3 HexNAc 3 NeuAc 1 +2OAc 1489.52 1 +20Ac-H 2 0 Hex 4 HexNAc 3 NeuAc 1 +2OAc 1651.58 1 +20Ac-H 2 0 Hex4HexNAc 4 NeuAc 1 +2OAc 1008.86 2 +20Ac-H 2 0 14 Table 3: Abbr. N- Molecular Calculated Observed Error tR No Name Copoion Formula Mass Mass (ppm) (min) Hex 4 HexNAc 3 Neu C61 H100 8.40, 1* Ac 1 +OAc 4_3_0_1+OAc N4045 1608.5660 1608.5637 -1.40 9.87 Hex 4 HexNAc 3 Neu 4_3_0_1+20A C63 H102 2* Ac 1 +20Ac c N4 046 1650.5765 1650.5662 -6.27 8.14 Hex 4 HexNAc 4 Neu C69 H113 7.68, 3* Ac 1 +OAc 4_4_0_1+0Ac N5 050 1811.6453 1811.6445 -0.48 8.42 7.68, 7.98, Hex 5 HexNAc 4 Neu C75 H123 8.72, 4 Ac1+0Ac 5_4_0_1+0Ac N5 055 1973.6982 1973.6993 0.60 9.40 Hex 4 HexNAc 5 Neu C77 H126 5* Ac 1 +0Ac 4_5_0_1+0Ac N6 055 2014.7247 2014.726 0.43 9.75 Hex 5 HexNAc 3 Neu C78 H127 6 Ac 2 +0Ac 5_3_0_2+0Ac N5 058 2061.7142 2061.7202 -2.53 6.65 8.75, Hex 5 HexNAc 4 dHex C81 H133 9.35, 7* 1 NeuAc 1 +OAc 5_4_1_1+OAc N5 059 2119.7561 2119.7595 -0.01 9.87 Hex 5 HexNAc 4 dHex 5_4_1_1+20A C83 H135 8.60, 8* 1 NeuAc 1 +20Ac c N5 060 2161.7666 2161.7697 1.42 9.20 7.93, 8.47, 8.80, Hex 5 HexNAc 4 Neu C86 H140 9.12, 9 Ac 2 +OAc 5_4_0_2+OAc N6 063 2264.7936 2264.7934 -0.08 9.40 Hex 5 HexNAc 4 Neu 5_4_0_2a+OA C86 H140 10* Ac 1 NeuGc 1 +OAc c N6 064 2280.7885 2280.7874 -0.50 9.02 8.34, 8.94, Hex 5 HexNAc 4 Neu 5_4_0_2+20A C88 H142 9.52, 11 Ac 2 +2OAc c N6 064 2306.8042 2306.8022 -0.83 10.12 Hex 5 HexNAc 5 dHex C89 H146 12* 1 NeuAc 1 +OAc 5_5_1_1+OAc N6 064 2322.8354 2322.8427 3.11 9.15 9.19, 9.39, 9.64, 9.80, 10.04, Hex 5 HexNAc 4 Neu 5_4_0_2+30A C90 H144 10.22, 13 Ac 2 +3OAc c N6 065 2348.8147 2348.8135 -0.50 10.41 Hex 5 HexNAc 5 dHex 5_5_1_1+20A C91 H148 9.48, 14 1 NeuAc 1 +20Ac c N6 065 2364.8460 2364.8431 -1.25 10.10 8.39, 8.69, 8.97, 9.20, 9.70, 9.85, Hex 5 HexNAc 4 dHex C92 H150 10.37, 15 1 NeuAc 2 +OAc 5_4_1_2+OAc N6 067 2410.8515 2410.8511 -0.14 10.64 Hex 5 HexNAc 4 dHex 1 NeuAc 1 NeuGc 1 +O 5_4_1_2a+OA C92 H150 8.67, 16* Ac c N6 068 2426.8464 2426.8349 -4.75 8.89 17* Hex 5 HexNAc 4 dHex 5_4_1_2+20A C94 H152 2452.8620 2452.8623 0.12 9.35, 15 1NeuAc 2 +20Ac c N6 068 9.57, 9.94, 10.42, 10.84, 11.59 Hex 5 HexNAc 5 Neu C94 H153 18* Ac 2 +0Ac 5_5_0_2+OAc N7 068 2467.8729 2467.8555 -7.06 11.02 9.82, 10.00, 10.31, Hex 5 HexNAc 4 dHex 5_4_1_2+30A C96 H154 10.49, 19* 1 NeuAc 2 +30Ac c N6 069 2494.8926 2494.8699 -1.10 10.67 8.85, 9.25, 9.75, Hex 5 HexNAc 4 Neu C97 H157 10.00, 20 Ac 3 +0Ac 5_4_0_3+0Ac N7 071 2555.8890 2555.8877 -0.51 10.46 9.05, 9.77, 10.19, 10.57, Hex 5 HexNAc 4 Neu 5_4_0_3+20A C99 H159 11.19, 21 Ac 3 +20Ac c N7 072 2597.8996 2597.9010 0.57 11.77 9.94, 10.27, 10.79, Hex 5 HexNAc 4 Neu 5_4_0_3+30A C1O H161 11.31, 22 Ac 3 +30Ac c N7 073 2639.9101 2639.9032 -2.61 12.01 C103 H167 9.85, Hex 5 HexNAc 4 dHex N7 075 10.22, 23 1NeuAc 3 +0Ac 5_4_1_3+0Ac 2701.9469 2701.9497 1.02 10.84 10.07, C105 H169 10.32, N7 07610.51, Hex 5 HexNAc 4 dHex 5_4_1_3+20A N7 07610.96, 24 1NeuAc 3 +2OAc c 2743.9575 2743.9537 -1.39 11.47 10.32, C107 H 171 10.79, N7 07711.11, Hex 5 HexNAc 4 dHex 5_4_1_3+30A N7 07711.59, 25 1NeuAc 3 +3OAc c 2785.9680 2785.9679 -0.04 12.46 10.0, C1II H180 10.37, N8 08110.81, Hex 6 HexNAc 5 Neu N8 08111.27, 26 Ac 3 +OAc 6_5_0_3+OAc 2921.0212 2921.0160 -1.77 11.46 10.32, C113 H182 10.72, N8 08211.02, Hex 6 HexNAc 5 Neu 6_5_0_3+20A N8 08211.44, 27 Ac 3 +2OAc c 2963.0318 2963.0250 -2.28 12.07 Hex 6 HexNAc 5 Neu 6_5_0_3+30A C115 H184 28 Ac 3 +3OAc c N8 083 3005.0423 3005.0390 -1.10 11.42 Hex 6 HexNAc 5 Neu 6_5_0_4+20A C124 H199 11.29, 29 Ac 4 +2OAc c N9 090 3254.1272 3254.1221 -1.57 11.77 Hex 6 HexNAc 5 Neu 6_5_0_4+30A C126 H201 11.49, 30 Ac 4 +3OAc c N9 091 3296.1378 3296.1379 0.06 11.96 a, two sialic acids, NeuAc and NeuGc, respectively; *, novel N-glycan. 16 Example 7 - Comparison of the N-glycomic profiles of rat, mouse, and human sera Serum N-glycans were divided into five classes: high mannose (high Man) N-glycans; undecorated complex/hybrid (C/H) N-glycans; fucosylated complex/hybrid (C/H-F) N glycans; sialylated complex/hybrid (C/H-S) N-glycans; and fucosylated-sialylated complex/hybrid (C/H-FS) N-glycans. Using the established glycomic approach, N-glycans of mouse and human sera were also comprehensively profiled and compared (FIG. 6A). Relative abundances were assigned to each glycan class on the basis of the total ion counts for the individual N-glycan signals (FIG. 6B). Sialylated N-glycans were much more abundant than neutral N-glycans in all three species, accounting for about 7 0

-

8 0 % of the total N-glycans in the serum of each species. Di-sialylated glycans were always the most dominant species, accounting for more than 50% of the total glycans, followed by mono-, tri-, and tetra-sialylated N-glycans (FIG. 7A). Fucosylated N-glycans (25-40%) were significantly less abundant than their counterparts (60-75%) in the sera of all three species (FIG. 7B), and more than 80% of the N-glycans in rat, mouse, and human sera possessed di antennary structures, mono- and tri-antennary structures were less abundant (FIG. 7C). Despite similarities in the total levels of N-glycans in sera among the three species, the differences in the composition of serum N-glycans were remarkable among rats, mice, and humans. First, the most notable difference in glycans among rat, mouse, and human sera was the type of sialic acid present (FIG. 8). In humans, sialic acids in the serum N-glycans were exclusively in the form of NeuAc, whereas NeuGc was the sole sialic acid moiety in mouse serum. However, both NeuAc and NeuGc were identified in the N-glycans of rat serum, in which the content of NeuAc was almost 20 times that of NeuGc. Uncommon 'mixed' N glycans possessing both NeuAc and NeuGc in one molecule were found solely in rat serum. Second, a large number of sialylated N-glycans with O-acetylation were detected in rat serum, which, by contrast, were of low abundance in human serum and extremely low abundance in mouse serum (FIG. 9). Our results indicated a species-specific composition of sialic acid in the N-glycans of human, mouse and rat sera. However, the glycomic profile of rat serum was more similar to that of human serum based on both the high abundance of NeuAc and similar glycomic distributions (FIG. 6). INDUSTRIAL APPLICABILITY The method of the present invention relates to identifying and characterizing rat serum N glycome for the first time using microfluidic chip-LC-ESI-QTOF MS and M/MS techniques 17 for selection of the most appropriate animal model for pathological and pharmacological studies. This methodology could be applied in future glycomics studies, such as biomarker discovery. While the foregoing invention has been described with respect to various embodiments and examples, it is understood that other embodiments are within the scope of the present invention as expressed in the following claims and their equivalents. Moreover, the above specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety. 18

Claims (5)

1. A method for detection and identification of glycomic biomarkers, especially unknown biomarkers and those in low abundance, from serum of a mammal for selection of the most appropriate animal model for pathological and pharmacological studies for a subject, said method comprising the steps of: (1) sample preparation; (2) liquid chromatography - mass spectrometry; and (3) comparing glycomic profiles from the serum between the mammal and the subject, wherein the (1) sample preparation further comprises: (i) thermal denaturation in SDS-containing buffer with addition of DTT; (ii) digestion by N-glycosidase F (PNGase F); and (iii) purification by Hypercarb solid-phase extraction (SPE) cartridges; and wherein the (2) liquid chromatography - mass spectrometry further comprises a microfluidic porous graphitized carbon (PGC) chip-LC/MS with Mobile phase condition of 0.5% formic acid of pH 3.0 in water and acetonitrile.

2. The method of claim 1, wherein the glycomic biomarkers are N-glycans.

3. The method of claim 2, wherein comparison of serum N-glycans are divided into five classes comprising high mannose N-glycans, undecorated complex/hybrid N-glycans, fucosylated complex/hybrid N-glycans, sialylated complex/hybrid N-glycans, and fucosylated-sialylated complex/hybrid N-glycans.

4. The method of claim 1, wherein the mammal from which the serum is compared comprises rat serum when the subject is either human or mouse.

5. The method of claim 4, wherein the rat serum comprises 129 N-acetylneuramic acid and 59 N-glycolylneuramic acid which is more similar to human serum than mouse serum based on high abundance of N-acetylneuramic acid and similar glycomic distributions. 19