Identification of Glycoproteins Containing Specific Glycans Using a

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Identification of glycoproteins containing specific glycans using a lectin-chemical method Yan Li, Punit Shah, Angelo M. De Marzo, Jennifer VanEyk, Qianqian Li, Daniel W. Chan, and Hui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504304v • Publication Date (Web): 02 Apr 2015 Downloaded from http://pubs.acs.org on April 8, 2015

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Analytical Chemistry

1 3

2

Identification of glycoproteins containing specific glycans using a lectin-chemical 4 5

method 6 7 9

8

Yan Li1,3, Punit Shah1, Angelo M. De Marzo1, Jennifer Van Eyk2, Qianqian Li3, Daniel 10 1

W. Chan1, and Hui Zhang1 12 13 15

14

Department of 1Pathology and 2Medicine, Johns Hopkins University, Baltimore, 16 17

Maryland, United State, 21287 19

18 3

Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, 100101

20 21

Running title: Identification of glycoproteins containing specific glycans 2 23 24 25

Corresponding author: Hui Zhang, Department of Pathology, Johns Hopkins 26 27

University, 400 N. Broadway, Smith Building, Room 4011 1550, Baltimore, MD 28 29

21287. Phone: (443)287-2986; Email: [email protected] 30 31 32 3

ABBREVIATIONS 34 36

35

SPEG:

solid-phase extraction of N-linked glycopeptides

38

MS:

mass spectrometry

37 39

MS/MS: tandem mass spectrometry 40 42

41

HPLC:

high-performance liquid chromatography

4

AAL:

Aleuria aurantia lectin

SNA:

Sambucus nigra lectin

WGA:

wheat germ agglutinin

43

46

45

48

47 49 50 51 52 53 54 5 56 57 58 59 60

ACS Paragon Plus Environment


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ABSTRACT 5

4

Glycosylation is one of the most common protein modifications. Each glycoprotein 7

6

can be glycosylated at multiple glycosites, and each glycosites can be modified by 9

8

different glycans. Due to this heterogeneity of glycosylation, it has proven difficult to 10 1

study the structure-function relationship of specific glycans and their affected 12 13

glycoproteins. Here, we report a novel method for rapid and quantitative identification 14 15

of glycoproteins containing specific glycans. Lectin affinity isolations are followed by 16 17

chemical immobilization of the captured glycopeptides, allowing the identification of 18 19

glycoproteins containing specific glycans by subsequent mass spectrometry. The 20 21

application of the method should be useful to facilitate our understanding of how 2 23

changes in glycan associate with diseases, and to discover novel glycoproteins with 24 25

certain glycans that could serve as biomarkers or therapeutic targets. 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


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INTRODUCTION 5

4

Glycosylation of proteins is known to play an important role in various biological 7

6

processes, and aberrant glycosylation also plays an important role in a large number 9

8

of human diseases 10

[1-4]

. Since glycoproteins typically contain variable glycan

1

structures at each glycosylation site (glycosite) 12

[5-6]

, the glycans present at each

13

glycosite on the glycoproteins have therefore attracted great interest, and elucidation 14 15

of their precise nature would greatly advance our understanding of their role in health 16 17

and disease. However, there are currently no methods available that are sufficiently 18 19

specific and sensitive for the analysis of glycans at each glycosite in complex 20 21

samples. 2 23 24 25

Recently, two popular glycoprotein enrichment methods have been established: 26 27

affinity capture based on glycan-lectin interaction and Solid Phase Extraction of 28 29

Glycopeptides (SPEG). For example, Kaji et al. isolated N-linked glycoproteins and 30 31

glycopeptides using a lectin affinity capture method, which was followed by tandem 3

32

mass spectrometry (MS/MS)[7]. Several studies reported the use of lectins for the 34 35

characterization of glycoproteins in serum and clinical samples 36

[8-12]

. However, the

37

capture specificity of glycopeptides, which is defined as the percentage of identified 38 39

peptides containing consensus N-linked glycosylation motif (NXT/S sequence, while 40 41

X is any amino acids except P), is insufficient. Presumably, the low specificity of this 42 43

method is caused mostly by binding of non-glycopeptides within proteins to lectins 4 45

due to the low affinity of the lectin-glycan interaction. 46 47 48 49

Chemical 50

immobilization

methods,

such

as

solid-phase

extraction

of

51

glycosite-containing peptides (SPEG), allows for specific isolation and identification 52 53

of glycopeptides 54

[13-14]

. Glycans of the cis-diol groups are oxidized and covalently

5

coupled onto a solid surface using hydrazide chemistry. The non-glycopeptides that 56 57

are not coupled are removed, and the glycopeptides are subsequently released using 58 60

59

endoglycosidase PNGase F prior to analysis using MS/MS. It has been demonstrated that by using the SPEG method, the glycopeptides could be captured with high



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1 3

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specificity, and then identified in a rapid, sensitive, and reproducible manner 5

4

[15-19]

.

However, the SPEG method cannot provide any structural information about glycans, 7

6

since the glycans are removed from the peptides before MS detection. 8 9 10 1

Here, we developed a novel method that allows for identification of N-linked 12 13

glycosites using tandem mass spectrometry using lectin affinity capture followed by 14 15

SPEG (LecSPEG). For proof-of-principle, we applied this method to compare the 16 17

glycosite-containing peptides in the serum sample. We showed that this method was 18 19

able to identify and quantify those glycopeptides from glycoproteins interacting with 20 21

specific lectins with high specificity and sensitivity. 2 23 24 25

EXPERIMENTAL PROCEDURES 26 27

Materials and reagents 28 29

Aleuria aurantia lectin (AAL)-, Sambucus nigra lectin (SNA)-, and wheat germ 30 31

agglutinin (WGA)-conjugated agarose beads were purchased from Vector 32 3

Laboratories (Burlingame, CA). Tris (2-carboxythyl) phosphine (TCEP) was 34 35

purchased from Pierce (Rockford, IL). Sequencing-grade trypsin was from Promega 36 37

(Madison, WI). Peptide-N-glycosidase F (PNGase) was from ProZyme (San Leandro, 38 39

CA). Sodium periodate, hydrazide resin, and P6 desalting spin columns were from 40 41

Bio-Rad (Hercules, CA). C18 and MCX desalting columns were from Waters (Milford, 42 43

MS). The HPLC-mass spectrometry platform used in this study includes an Eksigent 4 45

2D nanoLC system (Dublin, CA) and a Thermo Scientific Orbitrap Velos mass 46 47

spectrometer (Waltham, MA). 48

Magic C-18 packing material was from Michrom

49

Bioresources (Auburn, CA), and all other chemicals were purchased from Sigma (St. 50 51

Louis, MO). 52 53 54 5

Serum samples 56 57

The serum specimens and relevant clinical information were obtained with informed 58 60

59

consent and with the approval of the Institutional Review Board of the Johns Hopkins University. To establish the method, pooled serum samples from healthy men were


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1 3

2

used. 4 5 7

6

Glycoprotein extraction using affinity capture by lectin-conjugated agarose beads 9

8

(Lec meothd) 1

10

Lectin-conjugated agarose beads (250 μl) were pre-washed for three times with 1 ml 12 13

Tris-HCl buffer (0.2 M Tris-HCl, pH 7.5). The beads were mixed with Tris-HCl buffer 14 15

for 5~10 min, and the buffer was removed following centrifugation at 6,000 x g for 5 17

16

min. Aliquot of serum pooled (25 μl) was diluted 1:10 in Tris-HCl buffer, then 18 19

incubated with the beads at 4ºC overnight with gentle shaking. The unbound 20 21

non-glycoproteins were removed by washing the beads with Tris-HCl buffer for three 23

2

times. Beads were then incubated with 100 μl lectin-specific elution buffers (100 mM 24 25

fucose in Tris-HCl buffer for AAL, 0.5 mM lactose in Tris-HCl buffer for SNA, and 0.8 26 27

M N-acetyl-D-Glucosamine in 0.2 M acetic acid for WGA) for 30 min with gentle 28 29

shaking to elute glycoproteins. The supernatant was collected and desalted on a P6 30 31

column. Glycoproteins were denatured, reduced, and digested with trypsin as 32 3

previously described 35

34

[13-14]

Trypsin was then deactivated by incubating the sample at

80ºC for 15 min and 0.5 μl of PNGase F was used to remove N-linked glycans from 36 37

the peptides. The peptides were desalted using a C-18 column, and the eluate was 39

38

dried using a SpeedVac and resuspended in 10 μl of 0.4% acetic acid per sample. 40 41 42 43

Glycopeptide extraction using affinity capture by lectin-conjugated agarose beads 4 45

(Lec+Lec method) 46 47

Glycoproteins were extracted from a 25-μl aliquot of serum pooled from healthy men 48 49

using the Lec method as described above. The eluted glycoproteins were digested 50 51

with trypsin. The peptides were then desalted with a C-18 column and incubated with 53

52

100 μl of pre-washed lectin-conjugated agarose beads for glycopeptide enrichment 54 5

using the same procedures for glycoprotein extraction described above. The eluted 56 57

glycopeptides were desalted using a C-18 column and the glycopeptides were 60

59

58

treated with 0.5 l of PNGase F to release N-linked glycans from the peptides. The peptides were desalted again and dried using a SpeedVac, before being



1 3

2

resuspended in 10 l of 0.4% acetic acid per sample. 4 5 7

6

N-linked glycopeptide extraction using the combination of lectin affinity capture and 9

8

SPEG method (LecSPEG method) 10 1

The peptides enriched by lectins were oxidized with 10 mM sodium periodate at 4ºC 12 13

for 1 hour in the dark. The oxidized peptides were purified using C18 desalting 14 15

columns to remove the oxidant and conjugated to hydrazide beads at room 16 17

temperature for 4 hours in 80% acetonitrile. Non-conjugated peptides were removed 19

18

by washing the resin for three times with 800 μL washing buffer (1.5 M NaCl, H2O, 20 21

and 100 mM of NH4HCO3). The N-linked glycosite-containing peptides were released 23

2

from the resin with 0.5 μL of PNGase F in 100 mM of NH4HCO3 and incubated at 24 25

37°C overnight. After the final purification using a MCX desalting column, the 26 27

peptides were dried by SpeedVac and resuspended in 10 µl of 0.4% acetic acid 28 29

solution for subsequent mass spectrometry analysis. 30 31 32 3

Analysis of N-linked glycosite-containing peptides using a HPLC-Orbitrap-MS 34 35

platform 36 37

Peptide identification by liquid chromatography tandem mass spectrometry 38 39

(LC-MS/MS) analysis was performed using an Orbitrap MS/MS (Thermo Fisher 41

40

Scientific) interfaced with a nanoLC system (Eksigent). A 5 μl sample of isolated 42 43

peptides was loaded onto the HPLC-Orbitrap-MS platform. The HPLC mobile phase 4 45

A were composed of 0.2% formic acid in HPLC-grade water, while phase B were 46 47

composed of 0.2% formic acid in HPLC grade acetonitrile. The mobile phase B was 49

48

increased from 5% to 40% over 90 min at 300 nl/min. A C18 column (75 μm ID x 10 51

50

cm, Magic C18 material, 5 μm, 100 Å) was used for peptide separation. Mass 52 53

analysis was performed using a data-dependent analysis of the top ten precursors 54 5

and a dynamic exclusion of 30 seconds. The data was acquired at 30,000 resolution 56 57

for the precursor scans and 7500 for MS/MS. Target values of 1e6 for MS and 1e5 for 58 60

59

MS/MS were set with maximum injection times of 100 and 300 milliseconds, respectively. Data was acquired used the Monoisotopic Precursor Selection (MIPS)


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1 3

2

method, with Predictive AGC enabled. 4 5 7

6

The acquired data was searched against the Homo sapiens taxonomy of the RefSeq 9

8

database (Version 40, 04/16/2010) using the Mascot (Matrix Science version 2.2.0) 10 1

search algorithm within Proteome Discoverer (Thermo Scientific version 1.1). The 12 13

data was searched with 2 missed cleavages allowed and a tolerance of 15 ppm on 14 15

the precursors and 0.02 Dalton on the fragment ions. Modifications allowed included 16 17

carbamidomethyl cysteines set to static and deamidation of asparagines and 18 19

oxidation of methionines set to variable. Data were searched against a decoy 20 21

database and filtered to a 1% false discovery rate (FDR). The dataset was further 2 23

filtered to ensure that all the identified peptides had Mascot ion scores greater than 24 25

50. 26 27 28 29

RESULTS 30 31

Detection of glycosite-containing peptides with special glycan motifs using lectin 32 3

affinity capture and chemical immobilization 34 35

To compare the LecSPEG method with the two lectin enrichment methods, 36 37

glycoprotein enrichment (Lec method) and affinity enrichment of glycoproteins 38 39

followed by enrichment of glycopeptides from the enriched glycoproteins using lectins 40 41

(Lec + Lec method), we applied these three methods to aliquots from the same 42 43

pooled sample of human sera to determine the number of identified peptides (Figure 4 45

1), the number of identified glycopeptides, and the specificity of identified 46 47

glycopeptides to total peptides. 48 49 50 51

First, we employed the lectin SNA-conjugated agarose beads to capture the 52 53

glycoproteins by using the Lec method. A total number of 85 peptides were identified 54 5

from the serum sample, with only 2 peptides containing N-linked glycosylation sites 56 57

with NXT/S motif. The low capture specificity (2%) of glycosite-containing peptide 58 60

59

identified by this method suggests that it is not suitable for glycosite analysis (Supplementary Table 1).



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1 2 3 5

4

Next, lectin SNA-conjugated agarose beads were first used to capture glycoproteins 7

6

from the serum sample, and the isolated glycoproteins were digested to peptides. 9

8

The glycopeptides were then further isolated by lectin SNA-conjugated agarose 10 1

beads and analyzed by LC-MS/MS. Using this tandem lectin affinity capture method, 12 13

14 glycosite-containing peptides were identified from the same serum sample with 14 15

the percentage of specificity or identification of glycosite-containing peptides at 36%. 16 17

Thus, 18

the

Lec

+

Lec

procedure

increased

the

number

of

identified

19

glycosite-containing peptides and capture specificity when compared with the Lec 20 21

method (Supplementary Table 1). 2 23 24 26

25

Finally, LecSPEG was used to analyze glycoproteins from the same serum sample. 27 28

After isolation of glycopeptides by Lec + Lec method. They were captured by SPEG 29 31

30

method and analyzed by LC-MS/MS. We were able to identify 46 unique 32 34

3

glycosite-containing peptides from the serum sample with the capture specificity of 35 36

82% (Supplementary Table 1). Clearly the lecSPEG method has the power to identify 37 39

38

the most glycosites and has the highest specificity for the capture in crude serum 40 42

41

among the three methods tested. 43 4 45 47

46

To determine if the LecSPEG method possesses a principal advantage for 49

48

glycopeptide detection with specific glycan motifs, we compared the three methods 51

50

using SNA, AAL, and WGA-conjugated beads for glycopeptide identification. SNA 53

binds to glycans containing α-2,6-linked sialic acid; AAL binds to glycans containing 5

54

52

fucose; WGA binds to glycans containing terminal β-N-acetylglucosamine or sialic 57

56

acid 60

59

58

[20]

. In each case, more glycopeptides were identified with higher capture

specificity using LecSPEG method (Supplementary Table 1).


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Furthermore, we tested the reproducibility of the LecSPEG method for glycopeptide 5

4

analysis. The glycopeptides were isolated in quadruplicates from 25-μl serum 7

6

samples by LecSPEG method using SNA-conjugated agarose beads. Using this 9

8

hybrid method, 42 glycopeptides were identified in all four replicate samples (Figure 2, 10 1

Supplementary Table 2). The CVs of the peak intensities of the 42 glycopeptides 12 13

ranged from 2.1% to 39.1%. Only four glycopeptides displayed CVs greater than 30%, 14 15

and the majority of glycopeptides (38 of 42, 90.5%) had CVs lower than 30%, which 16 17

indicates that this multi-step method was able to generate reproducible results. 18 19 20 21

DISCUSSION 2 23

The interactions between lectins and glycans are relatively weak, and a large 24 25

proportion of peptides captured by lectins are non-glycopeptides. When we 26 27

sequentially combined lectin affinity capture with the SPEG method, the capture 28 29

specificity of glycopeptides was significantly improved from 2% (Lec) to 36% 30 31

(Lec+Lec), to further 82% (LecSPEG), while the number of identified unique 32 3

glycosites increased from 2 (Lec) to 14 (Lec+Lec), to 46 (LecSPEG) using 34 35

SNA-conjugated agarose beads. Similar results were also observed when using 36 37

more lectins for glycopeptide capture, including AAL and WGA (Supplementary Table 38 39

1). 40 41 42 43

In addition to its high specificity and sensitivity, the reproducibility of the method for 4 45

glycopeptide analysis is important, especially for clinical research, which usually 46 47

requires sample comparison. For the tandem SNA lectin and SPEG capture methods, 48 49

the CVs of most identified glycopeptides were lower than 30%, which is comparable 50 51

to those of tryptic peptides analyzed by LC-MS 52

[19, 21-22]

. Thus, our data indicate that

53

our system is feasible for quantification analysis of clinically samples. 54 5 56 57

Moreover, the glycan motif information can be obtained for each glycosite-containing 58 60

59

peptide using LecSPEG detection. The glycosite-containing peptides identified by SNA-LecSPEG method contained sialic acids (α2-6) on glycans. The glycopeptides



1 3

2

identified by AAL-LecSPEG and WGA-LecSPEG contained fucose, and terminal 5

4

β-N-acetylglucosamine or sialic acids, respectively. When the serum samples were 7

6

profiled using LecSPEG method, the glycan motif information and quantification data 9

8

based on LC-MS peak intensity clearly showed the heterogeneity of glycans at each 10 1

glycosite, that individual glycoproteins could contain multiple glycosites and each 12 13

glycosite has its own glycan motif patterns (Supplementary Table 3). For example, 14 15

the glycosite DFVnASSK of Heparin cofactor II protein contains glycan motifs of 17

16

fucose and terminal β-N-acetylglucosamine that bound to lectins AAL and WGA, 18 19

respectively (Supplementary Table 3). We can determine the relative quantities of 20 21

specific glycan motif at each glycopeptides from individual samples by directly 2 23

comparing the LC-MS data between different samples. 24 25 26 27

In summary, we developed a lectin affinity followed by SPEG capture method for 28 29

glycopeptide analysis, and tested the analytical performance of the LecSPEG in this 30 31

study. We demonstrated that our method can be used to profile glycopeptides with 32 3

specific glycan motifs. The newly developed LecSPEG method will be a useful tool 34 35

for subsequent glycoproteomic studies in both biological and clinical studies. 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


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ACKNOWLEDGEMENTS 5

4

This work was supported from Early Detection Research Network (NIH/NCI/EDRN) 7

6

grant U01CA152813 (H.Z.) and U24CA115102 (D.W.C), the Patrick C. Walsh 9

8

Prostate Cancer Research Fund (H.Z.), the United States Department of Defense 10 1

grant PC081386 (Y.L.), the National Natural Science of China, grant 31270909 (Y.L.), 12 13

and the Outstanding Technical Talent Award of Chinese Academy of Sciences (Y.L.). 14 15

We would like to thank Xiaer Sun and Helen Fedor for technical assistance, Dr 16 17

Robert Cole, Robert O'Meally, and Tatiana Boronina for assistance in mass 18 19

spectrometry and data analyses, Torsten Juellch for critical reading of the manuscript. 20 21

Serum samples were obtained from The Brady Urological Research Institute in Johns 2 23

Hopkins University, which is funded by NIH/NCI Prostate SPORE (P50CA58236). 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60



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1 Spiro, R.G. Glycobiology 2002, 12, 43R-56R. 7

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2 Drake, P.M.; Cho, W.; Li, B.; Prakobphol, A.; Johansen, E.; Anderson, N.L.; Regnier, 8 9

F.E.; Gibson, B.W.; Fisher, S.J. Clin Chem 2010, 56, 223-236. 10 1

3 Dennis, J.W.; Granovsky, M.; Warren, C.E. Bioessays 1999, 21, 412-421. 12 13

4 Durand, G.; Seta, N. Clin Chem 2000, 46, 795-805. 14 15

5 Schachter, H. Glycobiology 1991, 1, 453-461. 16 17

6 Neumann, U.; Brandizzi, F.; Hawes. C. Ann Bot 2003, 92, 167-180. 18 19

7 Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.; 20 21

Takahashi, N.; Isobe, T. Nat Biotechnol 2003, 21,667-672. 2 23

8 Drake, R.R.; Schwegler, E.E.; Malik, G.; Diaz, J.; Block, T.; Mehta, A.; Semmes, O.J. 24 25

Mol Cell Proteomics 2006, 5, 1957-1967. 26 27

9 Calvano, C.D.; Zambonin, C.G.; Jensen, O.N. J Proteomics 2008, 71, 304-317. 28 29

10 McDonald, C.A.; Yang, J.Y.; Marathe, V.; Yen, T.Y.; Macher, B.A. Mol Cell 30 31

Proteomics 2009, 8, 287-301. 32 3

11 Narimatsu, H.; Sawaki, H.; Kuno, A.; Kaji, H.; Ito, H.; Ikehara, Y. FEBS J 2010, 277, 34 35

95-105. 36 37

12 Ito, S.; Hayama, K.; Hirabayashi, J. Methods Mol Biol 2009, 534, 195-203. 38 39

13 Zhang, H.; Li, X.J.; Martin, D.B.; Aebersold, R. Nat BiotechnoL 2003, 21, 660-666. 40 41

14 Tian, Y.; Zhou, Y.; Elliott, S.; Aebersold, R.; Zhang, H. Nat Protoc 2007, 2, 42 43

334-339. 4 45

15 Pan, S.; Zhang, H.; Rush, J.; Eng, J.; Zhang, N.; Patterson, D.; Comb, M.J.; 46 47

Aebersold, R. Mol Cell Proteomics 2005, 4, 182-190. 48 49

16 Zhang, H.; Yi, E.C.; Li, X.J.; Mallick, P.; Kelly-Spratt, K.S.; Masselon, C.D.; Camp, 50 51

D.G.; Smith, R.D.; Kemp, C.J.; Aebersold R. Mol Cell Proteomics 2005, 4, 144-155. 52 53

17 Zhou, Y.; Aebersold, R.; Zhang, H. Anal Chem 2007, 79, 5826-5837. 54 5

18 Zhang, H.; Liu, A.Y.; Loriaux, P.; Wollscheid, B.; Zhou, Y.; Watts, J.D.; Aebersold, 56 57

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19 Li,Y.; Sokoll, L.J.; Rush, J.; Meany, D.; Zou, N.; Chan, D.W.; Zhang, H. Proteomics: Clinical Applications 2009, 3, 597-608.


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20 Tao, S.C.; Li, Y.; Zhou, J.; Qian, J.; Schnaar, R.L.; Zhang, Y.; Goldstein, I.J.; Zhu, 5

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H.; Schneck, J.P. Glycobiology 2008, 18, 761-769. 7

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21 Hawkridge, A.M.; Wysocky, R.B.; Petitte, J.N.; Anderson, K.E.; Mozdziak, P.E.; 8 9

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22 Nagaraj, N.; Mann, M. J Proteome Res 2011,10, 637-645. 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60



1 3

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FIGURE LEGENDS 5

4

Figure 1. Schematic diagram of the analytical procedure of glycosite detection using 7

6

a lectin affinity capture method combined with chemical immobilization. 9

8

Figure 2. The reproducibility of glycopeptides captured by LecSPEG method using 10 1

SNA conjugated agarose beads in serum samples in quadruplicate. 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


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FIGURES 5

4

Figure 1. 6 7 8 10

9 Glycoproteins enriched by lectin

13

12

1

Glycoproteins enriched by lectin


14 15 16 18

17 Proteolysis

19

Proteolysis

Proteolysis

20 21 2 23 24 PNGase F

25 26

Glycopeptides enriched by Lectin

Glycopeptides enriched by lectin

27 28 29 30

PNGase F

31

SPEG

3

32 MS Lec

35

34 36 37

PNGase F

38 39 MS Lec+Lec

41

40 42 43

MS LecSPEG

4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60



1 3

2

Figure 2 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


Page 16 of 17

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1 2 3 For TOC only

4 6

5 Glycoproteins enriched by lectin



7 Proteolysis

9

8

PNGase F

10

Proteolysis

Glycopeptides enriched by Lectin

PNGase F

1

Proteolysis

Glycopeptides enriched by lectin

SPEG

12

MS Lec PNGase F

13

MS Lec+Lec MS LecSPEG

14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


Identification of Glycoproteins Containing Specific Glycans Using a

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