Site-Specific Profiling of Serum Glycoproteins ... - ACS Publications

Subscriber access provided by UNIV OF DURHAM

Site-specific Profiling of Serum Glycoproteins Using Nlinked Glycan and Glycosite Analysis Reveals Atypical NGlycosylation Sites on Albumin and Alpha-1B-glycoprotein Shisheng Sun, Yingwei Hu, Li Jia, Shadi Toghi Eshghi, Yang Liu, Punit Shah, and Hui Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Site-specific Profiling of Serum Glycoproteins Using N-linked Glycan and Glycosite Analysis Reveals Atypical N-Glycosylation Sites on Albumin and Alpha-1B-glycoprotein

Shisheng Sun1,2*, Yingwei Hu2, Li Jia1, Shadi Toghi Eshghi2, Yang Liu2, Punit Shah2, and Hui Zhang2

1

Department of Life Science, Northwest University, Xi’an, Shaanxi province 710069, China 2

Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21287, USA.

*Corresponding author: Shisheng Sun at [email protected] and Hui Zhang at [email protected].

ACS Paragon Plus Environment

1

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

ABSTRACT

Most serum proteins are N-linked glycosylated and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum Nglycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked Glycans And Glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by at least two glycans, with a majority of them were sialylated. Specifically, 3/4 of glycosites were modified by biantennary N-glycans and 1/3 of glycosites were modified by triantennary sialylated N-glycans. In addition, two novel atypical glycosites (with N-X-V motif) were identified and validated from albumin (ALB) and Alpha-1Bglycoprotein (A1BG). The widespread of these two glycosites among individuals were further confirmed by individual serum analyses.


2

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION

Serum is one of the most valuable clinical sample sources for disease diagnostics and therapeutic monitoring.1 It contains not only the major plasma protein constituents, such as albumin, immunoglobulins, transferrin, haptoglobin, and lipoproteins, but also many other proteins that are synthesized and secreted, shed, or lost from various cells and tissues.2 The alteration of serum protein concentrations and post-translational modifications (PTMs) could potentially reflect the physiological and pathological status of individuals. Therefore, characterization of serum proteins as well as their post-translational modifications (PTMs) is an essential prerequisite for their clinical utilities. A large amount of serum proteins are N-linked glycoproteins.3 Glycoproteins play important roles in various biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death.4-7 Aberrant glycosylation is usually associated with the pathological progression of many diseases.8 Many serum glycoproteins have been used as biomarkers for diagnosis and monitoring of disease progression, such as PSA for prostate cancer, AFP for liver cancer, CA-125 for ovarian cancer, as well as CA-199 and CEA for colon cancer.9 However, most of clinical tests measure serum glycoproteins at their expression level, while detailed glycosylation information could provide additional information for the studies of disease mechanisms and disease detection.10 It has been reported in previous studies, some distinctive glycoforms on specific glycoproteins could enhance the specificity and/or sensitivity of cancer diagnosis. For example, fucosylated AFP (AFP-L3), which is able to distinguish hepatocellular carcinoma (HCC) from benign liver diseases, has been used as a HCC biomarker.10 The fucosylation of glycans on GP73, kallikrein and haptoglobin also showed great potentials for


3


Page 4 of 29

early detection of HCC as well as disease progression monitoring.11 Therefore, comprehensive glycoproteome profiling of serum is very important for its clinical studies.

During the last decade, thousands of N-linked glycoproteins have been identified from human serum by mass spectrometry based approaches.2,3,12-21 However, these studies focused on deglycosylated peptides (or released glycans), but lack the site-specific glycosylation information. In this study, we used solid-phase extraction of N-linked Glycans And Glycositecontaining peptides method (NGAG)22, tandem mass spectrometry, and GPQuest software23 to profile serum N-glycoproteome at site-specific glycosylation level in addition to identification of glycans and glycosites. Especially, two atypical glycosylation sites from albumin (ALB) and Alpha-1B-glycoprotein (A1BG) were identified from serum and verified from individual blood serum, revealed their widespread existence.

EXPERIMENTAL SECTION

Human sera

Human sera (pooled from >100 individuals) used for comprehensive glycosylation profiling were purchased from Sigma (S7023). Individual sera from 16 donors were obtained from The Johns Hopkins Hospital according to the Institutional Review Board strictures of the Johns Hopkins University. All sera were stored at -80°C till use.

Protein digestion

Serum proteins (~75mg/mL) were denatured in 8M urea/1M NH4HCO3 buffer, reduced by 10mM TCEP at 37°C for 1h and alkylated by 15mM iodoacetamide at room temperature in the


4

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dark for 30 min. The solutions were diluted 5-fold with deionized water. Sequencing grade trypsin (Promega, Madison, WI; protein: enzyme, 40:1, w/w) was then added to the samples and incubated at 37°C overnight with shaking. Samples were centrifuged at 12,000g for 10min to remove any particulate matter and then purified by a C18 column (Waters, Milford, MA). Peptides were eluted from the C18 column in 60% ACN/0.1% TFA and the peptide concentrations were measured by BCA reagent (Thermo Fisher Scientific, Fair Lawn, NJ).

Extraction of N-linked glycans and glycosite-containing peptides using the NGAG method

Four milligram of peptides from pooled sera were (1mL) used for extraction of N-linked glycans and glycosite-containing peptides using the NGAG method. The guanidination of lysine on tryptic peptides was performed by adding 1mL of 2.85M aqueous ammonia and 1mL of 0.6M omethylisourea (Sigma-Aldrich, St. Louis, Missouri) into 1mL of peptide solution (final pH=10.5) and incubating at 65°C for 30min24. The reaction was stopped by adding 3mL of 10% TFA (make sure the solution pH100 individuals) by the NGAG method and identified using mass spectrometry. The enriched intact glycopeptides were then identified by searching GPQuest against identified glycans and glycosite-containing peptides from sera. (B) The glycan types identified from serum by the NGAG method. (C) The glycosites containing either N-X-S or N-X-T motif using the NGAG method.

Figure 2. The site-specific glycosylation profiling of human serum glycoproteins. (A) Heat map of identified intact glycopeptides from serum. The peptide-spectrum-matches (PSMs) of the intact glycopeptides, comprising of different glycans (upper) and glycosites (left) were exhibited in the heat map. The numbers of glycosites modified by each glycan and of glycans at each glycosite were summarized at the bottom and right parts of the figure, respectively. N: HexNAc; H: Hex; F: Fucose; S: Sialic acid. (B) The PSMs of different glycans with 0-4 sialic acids. (C) Top ten glycans detected on serum glycoproteins.

Figure 3. Identification and validation of an atypical N-glycosite on human serum albumin using site-specific

glycosylation

analysis.

(A)

A

spectrum

of

the

intact

glycopeptide

LV68N#EVTEFAK + HexNAc4Hex5Ac1 from albumin. (B) A spectrum of the intact glycopeptide LV68N#EVTEFAK + HexNAc4Hex5Ac2 from albumin. The oxonium ions (green) were used to extract the intact glycopeptide spectrum, the masses of the precursor and peptide/peptide+glycan fragment ions (orange) as well as the b/y-ions of the peptide portion (b-ions: blue; y-ions: red)


22

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were used for intact glycopeptide identification. (C) A spectrum of the deglycosylated form of the peptide LV68N#EVTEFAK.

68

Asn on the peptide was identified as the glycosylation site

based on the deamidation.

Figure 4. Estimation of glycosylation stoichiometries on the atypical glycosites Asn-68 from albumin (A) and Glycosite Asn-62 from A1BG (B). The glycosylation occupancies at the atypical glycosites were estimated by using the following formula:

ܲ௚௟௬௖௢ = (

‫ܣ‬ௗ௘௔௠௜ௗ௔௧௜௢௡_ଶ ‫ܣ‬ௗ௘௔௠௜ௗ௔௧௜௢௡_ଵ − ) × 100% ‫ܣ‬௡௢௡௚௟௬௖௢_ଶ + ‫ܣ‬ௗ௘௔௠௜ௗ௔௧௜௢௡_ଶ ‫ܣ‬௡௢௡௚௟௬௖௢_ଵ + ‫ܣ‬ௗ௘௔௠௜ௗ௔௧௜௢௡_ଵ

Wherein Pglyco represents the N-linked glycosylation occupancy, Adeamidation_1 and Anonglyco_1 represent the peak area of the same peptides with and without deamidation (N) in serum peptide samples, respectively. Adeamidation_2 and Anonglyco_2 represent the peak area of the same peptides with and without deamidation (N) in PNGase F treated serum peptide samples, respectively.

Figure 5. Widespread distribution of N-glycosylation at the atypical glycosites Asn-68 of albumin and Asn-62 of A1BG among population. The distribution of the intact glycopeptides from albumin and from A1BG among 16 individual serum are shown in Figure 4a and 4b, respectively. The total intensities of each sample were used for normalization by SEIVE (Thermo Fisher) and the intensities of the intact glycopeptides were used to exhibit their relative abundances among samples.


23


Figure 1. Profiling of N-linked glycans, glycosites, and intact glycopeptides from human sera. 117x134mm (300 x 300 DPI)


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. The site-specific glycosylation profiling of human serum glycoproteins. 211x130mm (300 x 300 DPI)



Figure 3. Identification and validation of an atypical N-glycosite on human serum albumin using site-specific glycosylation analysis. 120x54mm (300 x 300 DPI)


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Estimation of glycosylation stoichiometries on the atypical glycosites Asn-68 from albumin (A) and Glycosite Asn-62 from A1BG (B). 68x23mm (300 x 300 DPI)



Figure 5. Widespread distribution of N-glycosylation at the atypical glycosites Asn-68 of albumin and Asn-62 of A1BG among population. 58x20mm (300 x 300 DPI)


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC graphic 44x23mm (300 x 300 DPI)


Site-Specific Profiling of Serum Glycoproteins ... - ACS Publications

Recommend Documents