Enzymatic Tagging of Glycoproteins on the Cell Surface for Their

Subscriber access provided by Macquarie University

Article

Enzymatic Tagging of Glycoproteins on the Cell Surface for Their Global and Site-Specific Analysis with Mass Spectrometry Fangxu Sun, Suttipong Suttapitugsakul, and Ronghu Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00441 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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 30 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

Enzymatic Tagging of Glycoproteins on the Cell Surface for Their Global and Site-Specific Analysis with Mass Spectrometry

Fangxu Sun, Suttipong Suttapitugsakul and Ronghu Wu*

School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

1 ACS Paragon Plus Environment

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 30

ABSTRACT: The cell surface is normally covered with sugars that are bound to lipids or proteins. Surface glycoproteins play critically important roles in many cellular events, including cell-cell communications, cell-matrix interactions, and response to environmental cues. Aberrant protein glycosylation on the cell surface is often a hallmark of human diseases such as cancer and infectious diseases. Global analysis of surface glycoproteins will result in a better understanding of glycoprotein functions and the molecular mechanisms of diseases, and the discovery of surface glycoproteins as biomarkers and drug targets. Here, an enzyme is exploited to tag surface glycoproteins, generating a chemical handle for their selective enrichment prior to mass spectrometric (MS) analysis. The enzymatic reaction is very efficient and the reaction conditions are mild, which are well-suited for surface glycoprotein tagging. For biologically triplicate experiments, on average 953 N-glycosylation sites on 393 surface glycoproteins per experiment were identified in MCF7 cells. Integrating chemical and enzymatic reactions with MS-based proteomics, the current method is highly effective to globally and site-specifically analyze glycoproteins only located on the cell surface. Considering the importance of surface glycoproteins, this method is expected to have extensive applications to advance glycoscience.


Page 3 of 30 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

Surface glycoproteins are essential for cells and they regulate many cellular events, including cell-cell communication, cell signaling and immune defense.1-2 Aberrant glycosylation is often correlated with human diseases.3-4 Besides its impacts on cell adhesion and migration, changes in glycosylation of receptors on the cell surface can alter cell signaling and help tumor cells escape from immune surveillance.2, 5 Therefore, surface glycoproteins can serve as effective biomarkers for cancer diagnosis.6-7 Furthermore, glycoproteins located on the cell surface may serve as drug targets for disease treatments, especially when macromolecules such as antibodies or enzymes are developed as drugs in promising immunotherapy.8-9 Despite the importance of surface glycoproteins, the analysis of the surface glycoproteome is underrepresented compared to the whole proteome analysis because it is extraordinarily challenging to comprehensively analyze glycoproteins only located on the cell surface. Besides the low abundance of many glycoproteins and the heterogeneity of glycans, it is also difficult to specifically target surface glycoproteins. Antibody-based analysis has provided valuable information for cell surface glycoproteins.10 However, the low throughput and high cost have limited their wide applications. Technological advancements in mass spectrometry (MS) provide an opportunity to globally identify and quantify proteins and their modifications.11-21 In combination with MS, subcellular fractionation for the plasma membrane has been employed to analyze cell surface proteins,22 but it is not specific and the contaminants from highly abundant intracellular proteins are a serious issue. The combination of lectin enrichment and MS has provided another powerful method for glycoprotein analysis,23-24 but unfortunately it cannot be applied for surface glycoprotein analysis. 3 ACS Paragon Plus Environment


Page 4 of 30

Several years ago, Wollscheid et al. developed an elegant method, termed cell surface capturing (CSC) technology, to systematically analyze surface N-glycoproteins.25 This technique uses sodium periodate (NaIO4) to generate a chemical reporter on the extracellular glycan moieties which can be used for surface glycoprotein enrichment prior to MS analysis. Recently, bioorthogonal chemistry offers another excellent opportunity to analyze cell surface glycoproteins,26-28 in which a sugar analog with a chemical reporter is incorporated into glycoproteins through the biosynthetic machinery of a cell.29 Previously, we combined metabolic labeling, copper-free click chemistry, enzymatic reaction and MS-based proteomics to globally and site-specifically analyze cell surface N-glycoproteins.30-34 Strain-promoted alkyne−azide cycloaddition (SPAAC),35 which avoids cytotoxic copper ions, is very mild and suitable for tagging cell surface glycoproteins. However, this method relies on metabolic labeling, and thus its applications to tissue or clinical samples are restricted. Enzymatic-based methods have great potential to tag surface glycoproteins because normally the reaction conditions are mild and the reactions are highly efficient.36 In this work, global and site-specific analysis of N-glycoproteins on the cell surface was achieved through an effective method integrating enzymatic and chemical reactions with MSbased proteomics. Galactose oxidase (GAO), which specifically converts the hydroxyl group at C6 of Gal/GalNAc to the aldehyde group, was employed to tag cell surface glycoproteins. In order to make the oxidation more efficient, horseradish peroxidase (HRP) was added, which shifts the reaction towards completion by consuming one of the products, i.e. H2O2, and activates the deactivated form of GAO. The enzymatic reaction conditions are mild, which can minimize the stress posed on cells, and thus maintain the intact cellular state. After cell lysis, surface glycoproteins containing the aldehyde group were enriched with the hydrazide beads, followed


Page 5 of 30 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


by on-bead digestion and the removal of non-glycopeptides. Glycopeptides were released through hydrazine-oxime exchange, and the enriched surface glycopeptides were treated with peptide N-glycosidase F (PNGase F) in heavy-oxygen water. The release of glycopeptides from the hydrazide beads eliminates the possible steric hindrance during the PNGase F treatment. Finally, a unique tag was generated on the N-glycosylation sites for MS analysis. When neuraminidase was utilized to remove the terminal sialic acids on glycans, more surface glycoproteins were identified. The experimental results demonstrated that the current method is highly effective to cover cell surface glycoproteins and can be applied for comprehensive and site-specific analysis of surface glycoproteins.

EXPERIMENTAL SECTION

Cell Culture MCF7 cells (from American type culture collection (ATCC)) were grown in Dulbecco's modified eagle's medium (DMEM) (Sigma-Aldrich) containing high glucose and 10% fetal bovine serum (FBS) (Thermo) inside a humidified incubator with 5.0% CO2 at 37 °C. Jurkat cells (ATCC) were grown in Roswell Park Memorial Institute (RPMI) 1640 media (Sigma) with 10% FBS. For the quantification experiment, cells were grown in DMEM with 10% dialyzed FBS (Corning) supplemented with 0.146 g/L

13

C615N2 L-lysine (Lys-8) and 0.84 g/L

13

C6 L-

arginine (Arg-6) (Cambridge Isotopes Inc.) or lysine (Lys-0) and arginine (Arg-0) with the same



Page 6 of 30

concentrations. After cells were cultured for at least six generations, heavy cells were treated with BFA (5 µg/mL) for 18 hours and the light cells were treated with DMSO as a control.

Oxidation of Glycans with GAO and Removal of Sialic Acid with Neuraminidase When the confluency of cells reached about 80%, cells were washed twice with phosphate buffered saline (PBS, Sigma) and harvested by scraping. The cells were pelleted by centrifugation at 300 g for 3 minutes and washed once with cold PBS. Then cells were oxidized with a PBS solution containing galactose oxidase (50 U/mL, Innovative Research), HRP (40 U/mL, Sigma) and 5% FBS for 1 h at 37 °C. For the removal of sialic acids, cells were treated with neuraminidase (250 mU/mL, Sigma) in PBS (pH=6.7) at 37 °C for 30 min before harvest, followed by the oxidation of surface glycoproteins with GAO.

Enrichment of Surface Glycoproteins and Treatment with PNGase F After cell lysis, the cell lysate was centrifuged, and the resulting supernatant was transferred to a new tube. Hydrazide beads (100 μL, Thermo Scientific) were washed three times with deionized water and transferred to the tube with the supernatant from cell lysis. After adding aniline (10 mM), the mixture was incubated at 4 °C through end-over-end rotation for 24 hours. On the next day, the beads were washed three times with a buffer containing 8 M urea, 0.4 M ammonium carbonate (NH4HCO3), and 0.1% sodium dodecyl sulfate (SDS). In this case, only tagged surface glycoproteins were enriched with the hydrazide beads. After on-bead digestion with trypsin, glycopeptides were eluted twice by incubating with the elution buffer (0.2 M methoxylamine hydrochloride, 1.5 M NaCl, and 0.1 M aniline in 0.1 M 6 ACS Paragon Plus Environment

Page 7 of 30 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


sodium acetate solution, pH 4.5) at 37 °C for 30 and 60 minutes, respectively. Purified and completely dried peptides were then dissolved in 40 μL buffer containing 50 mm NH4HCO3 (pH=9) in heavy-oxygen water (H218O) and deglycosylated with peptide-N-glycosidase F (PNGase F, Sigma-Aldrich) for 3 hours. The detailed information about protein alkylation and digestion, and the treatment with PNGase F is in Supporting Information.

LC-MS/MS Analysis, Database Search and Data Filtering The glycopeptide samples were dissolved in loading buffer consisting of 5% ACN and 4% FA, and loaded onto a microcapillary column packed with C18 beads. Peptides were separated by HPLC and detected in a hybrid dual-cell quadrupole linear ion trap-Orbitrap MS. LC-MS analysis is described in more details in Supporting Information. SEQUEST (version 28) was used to search glycopeptides after converting the raw files to an mzXML format.37 Spectra were matched against a database consisting of sequences from all human proteins (Homo sapiens) downloaded from the UniProt. The following parameters were employed for the database search: 20 ppm precursor mass tolerance; 1.0 Da product ion mass tolerance; fully digested with trypsin; up to three missed cleavages; variable modifications: oxidation of methionine (+15.9949 Da), 18O tag of Asn (glycosylation site) (+2.9883 Da), heavy lysine

(+8.0142

Da)

and

heavy

arginine

(+6.0201

Da);

fixed

modifications:

carbamidomethylation of cysteine (+57.0214 Da). The target-decoy method, in which each sequence from a protein was listed in both forward and reverse orders, was used to evaluate the false discovery rates (FDR) of glycopeptide and glycoprotein identifications.38 The quality of glycopeptide and glycoprotein identification



Page 8 of 30

was evaluated and controlled by linear discriminant analysis (LDA) employing multiple parameters such as XCorr, charge state, and precursor mass error.39 Peptides with fewer than seven amino acids were deleted and peptide spectral matches were filtered to

Enzymatic Tagging of Glycoproteins on the Cell Surface for Their

Recommend Documents