Letter pubs.acs.org/ac
Large-Scale Measurement of Absolute Protein Glycosylation Stoichiometry Shisheng Sun and Hui Zhang* Department of Pathology, Johns Hopkins University, 400 North Broadway, Smith Building, Room 4011, Baltimore, Maryland 21287, United States S Supporting Information *
ABSTRACT: Protein glycosylation is one of the most important protein modifications. Glycosylation site occupancy alteration has been implicated in human diseases and cancers. However, current glycoproteomic methods focus on the identification and quantification of glycosylated peptides and glycosylation sites but not glycosylation occupancy or glycoform stoichiometry. Here we describe a method for large-scale determination of the absolute glycosylation stoichiometry using three independent relative ratios. Using this method, we determined 117 absolute N-glycosylation occupancies in OVCAR-3 cells. Finally, we investigated the possible functions and the determinants for partial glycosylation.
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as the total protein (Figure 1, eqs 3 and 4). Therefore, by determining these three relative ratios using quantitative proteomics and glycoproteomics, the absolute glycosylation occupancy of the glycosite in different states can be calculated (Figure 1, eqs 5 and 6, as States I and II, respectively). On the basis of this principle, the stoichiometry of a given glycoform can also be determined by comparing the changes in relative abundance between the glycoform-containing and nonglycoform-containing forms of the glycosite. The method is therefore
rotein glycosylation plays important roles in biological processes, and aberrant glycosylation is usually associated with disease progression.1 The glycosylation level of glycoproteins at each glycosylation site can be affected by enzymes that are responsible for glycosylation synthesis, glycoprotein expression and structures, the availability of nucleotide sugars, as well as the nucleotide sugar transporters.2 For example, the occupancy at the glycosylation sites has been implicated in human diseases such as type-I congenital disorders of glycosylation (CDG-I)3 and cancers.4−6 Several glycoproteomic methods have been developed for large-scale characterization of glycoproteins and glycosylation sites.7−9 However, these methods focus on the identification and quantification of glycosylated peptides and glycosylation sites but not their glycosylation stoichiometry. Here, we show that the site-specific glycosylation stoichiometry of glycoproteins can be obtained using a high-throughput approach by comparing changes in relative abundance between occupied and unoccupied glycosites among the same glycoproteins from different conditions. The glycosylation occupancy determination using this method is based on the fact that the total percentage of glycosylated and nonglycosylated forms of a glycosylation site is 100% (Figure 1, eq 1, as State I). Therefore, when the glycosylation occupancy at a partially glycosylated site changes with different conditions in cells or organisms, the percentages of glycan occupied and unoccupied peptides at each specific glycosylation site can be altered (Figure 1, eq 2, as State II). The percentages of occupied and unoccupied glycosylation by specific glycans or all glycans can be calculated from the percentages in the original state using the three relative ratios, including the ratios of glycosylated and nonglycosylated forms of the glycosite as well © XXXX American Chemical Society
Figure 1. Principle of absolute glycosylation stoichiometry measurement. Pglyco1, percentage of a glycosylated peptide at state I; Pnonglyco1, percentage of the nonglycosylated form of the glycosite-containing peptide at state I; Pglyco2, percentage of a glycosylated peptide at state II; Pnonglyco2, percentage of the nonglycosylated form of the glycositecontaining peptide at state II; Rglyco, the state II/I ratio of the glycosylated peptide; Rnonglyco, state II/I ratio of the nonglycosylated form of the glycosite-containing peptide; Rprotein, state II/I ratio of the total protein. Received: May 3, 2015 Accepted: June 11, 2015
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DOI: 10.1021/acs.analchem.5b01679 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 2. Determination of absolute glycosylation occupancy in OVCAR-3 cells. (A) The workflow and strategy for absolute N-linked glycosylation occupancy measurement in OVCAR-3 cells. The OVCAR-3 cells were treated by tunicamycin (TM) to inhibit their N-linked glycosylation, and SILAC technology was used for accurate peptide and protein quantification. (B) The TM-treated/native ratios of the glycosylated and nonglycosylated forms of the glycosite-containing peptides as well as their proteins. (C) The glycosite occupancies in native and TM-treated cells (based on part b). (D) Distribution of glycosite occupancies in native and TM-treated OVCAR-3 cells. (E) Two examples of glycosite occupancy diversities in the same protein.
occupancy (>70%) sites, we found that only 10% of the sites had a low occupancy in untreated OVCAR-3 cells, while almost half of the sites had a low occupancy in TM-treated cells. Meanwhile, the highly occupied sites were reduced from 51% in native cells to 5% in TM-treated cells (Figure 2D). These results indicated that tunicamycin inhibited the overall glycosylation occupancies at these identified partial glycosylation sites. A total of 64% of the measured partially glycosylated proteins only contain one partial glycosylation site, whereas the remaining 36% of partially glycosylated proteins contain two to eight partially glycosylated sites (Figure S1 in the Supporting Information). The diversities of the glycosylation occupancies were observed among different glycosites of the same proteins. For example, among four partial glycosylation sites in Endoplasmin (HSP90B1 or GRP94), one of them had a low occupancy, one had a high occupancy, and another two had moderate occupancies in native OVCAR-3 cells (Figure 2E). In another example, among five partially glycosylated sites identified in Galectin-3-binding protein (LGALS3BP), only one has a low glycosylation occupancy while all other four are highly occupied in native OVCAR-3 cells (Figure 2E). To investigate whether the partially glycosylated proteins were involved in certain biological functions in the cells, we performed gene ontology (GO) and pathway analyses of these proteins. The GO and KEGG pathway analyses indicated that many partially glycosylated proteins were involved in protein and glycan biosynthesis in the ER and protein and glycan degradation in the lysosome (Figure 3A,B and Figure S2 in the Supporting Information). These partially glycosylated proteins in the ER and lysosome may have some functions for cell survival under various conditions. For example, Endoplasmin, also known as HSP90B1 or GRP-94, is a heat shock protein in the ER, and it functions as a molecular chaperone in the processing, folding, and transport of secreted proteins.11 It plays a very important role in ER quality control and assists the targeting of the unfolded or misfolded proteins for the ER associated degradation (ERAD) pathway.12 In our study, five
applicable for calculation of the stoichiometry of not only the overall glycosite occupancy but also a given glycoform such as sialylation, fucosylation, or a specific glycoform binding to a lectin. We first evaluated the method by using it to define the absolute stoichiometries of N-linked glycosylation in the OVCAR-3 cell line. In order to change the glycosite occupancies, we used a tunicamycin (TM) treatment strategy to inhibit the overall N-glycosylation occupancies of the cells (Figure 2A). Proteins from untreated cells labeled with heavy SILAC medium were mixed with the same amount of proteins from the TM-treated cells grown in light SILAC medium and were digested into peptides by trypsin. In total, 6 mg of peptides were used for isolation of glycosite-containing peptides (deglycosylated peptides) by using the SPEG method based on hydrazide chemistry,8,10 and enriched glycositecontaining peptides were fractionated into 12 fractions by bRPLC prior to liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis. The remaining peptides (∼200 μg) were also directly fractionated into 24 fractions followed by LC−MS/MS analysis for global proteomic analysis. The relative abundance changes of glycopeptides in TM-treated cells were quantified from glycoproteomic analysis, while the relative abundance changes of unoccupied glycosite-containing peptides and other tryptic peptides were quantified by global proteomic analysis (Figure 2A). By using three TM-treated/ native ratios (occupied glycosite-containing peptide, unoccupied glycosite-containing peptide, and the total protein) at each partially glycosylated site which was determined by simultaneously identifying both the occupied and unoccupied forms of the same glycosite-containing peptide (Figure 2B and Supplementary Table 1 in the Supporting Information), we determined the absolute occupancies at 117 partial glycosylation sites in the native and TM-treated OVCAR-3 cells (Figure 2C and Supplementary Table 2 in the Supporting Information). When we classify these measured partial glycosylation sites into low occupancy (
