Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

Abstract

Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O-glycosylation. Here, we demonstrate a substantial improvement in the SimpleCell strategy by including an additional stage of lectin affinity chromatography on secreted glycoproteins from culture media (secretome) and by incorporating pre-fractionation of affinity-enriched glycopeptides via IEF before nLC-MS/MS. We applied these improvements to three human SimpleCells studied previously, and each yielded a substantial increase in the number of O-glycoproteins and O-glycosites identified. We found that analysis of the secretome was an important independent factor for increasing identifications, suggesting that further substantial improvements can also be sought through analysis of subcellular organelle fractions. In addition, we uncovered a substantial nonoverlapping set of O-glycoproteins and O-glycosites using an alternative protease digestion (chymotrypsin). In total, the improvements led to identification of 259 glycoproteins, of which 152 (59%) were novel compared with our previous strategy using the same three cell lines. With respect to individual glycosites, we identified a total of 856 sites, of which 508 (59%) were novel compared with our previous strategy; this includes four new identifications of OGalNAc attached to tyrosine. Furthermore, we uncovered ϳ220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. The improved strategy should greatly facilitate high throughput characterization of the human GalNAc-type O-glycoproteome as well as be applicable to analysis of other O-glycoproteomes.

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

“…O-GalNAc was simplified by targeting the private chaperone, COSMC, for the core UDP-Gal: GalNAcα1-O-Ser/Thr β1,3gal-actosyltransferase (C1GalT1) controlling the first step in O-GalNAc elongation (16). We previously developed a workflow for isolation and characterization of GalNAc O-glycopeptides from O-GalNAc SimpleCells (16,27), which we modified for Man O-glycopeptides using Concanavalin A (Con A) lectin chromatography (Fig. 1B).…”

“…The conditions of LC analysis were essentially as previously described (27). The EASY-nLC II was equipped with a single analytical column set up using PicoFrit Emitters (New Objectives, 75-μm inner diameter) packed in-house with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 3-μm particle size).…”

“…Mass spectrometric analyses were performed essentially as previously described (27). Samples were analyzed either on a setup composed of an EASY-nLC II (Thermo Fisher Scientific) interfaced via a nanoSpray Flex ion source to an LTQ-Orbitrap XL hybrid spectrometer (Thermo Fisher Scientific) or an EASY-nLC 1000 (Thermo Fisher Scientific) interfaced via a nanoSpray Flex ion source to an LTQ-Orbitrap Velos Pro hybrid spectrometer (Thermo Fisher Scientific).…”

“…Data processing was carried out using Proteome Discoverer 1.4 software (Thermo Fisher Scientific) as previously described (27) with minor changes in preprocessing and processing procedures. The monosaccharide subtraction routine for correctly interpreting HCD spectra was used as previously described (27), i.e., the exact masses of 1, 2, 3, and 4 hexose (Hex) units were subtracted from the corresponding precursor ion mass, generating four distinct files. The .raw files and subtracted .mgf files were searched against the human-specific UniProt database downloaded on Feb. 13, 2013, using semispecific trypsin, chymotrypsin, or GluC proteolytic cleavage.…”

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

“…The column was washed with 10 column volumes (CVs) of ConA buffer A at 100 μL/min before elution with five CVs of ConA buffer B (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl 2 /MgCl 2 /MnCl 2 /ZnCl 2 , 0.5 M methyl-α-D-glucopyranoside/methyl-α-D-mannopyranoside) at 50 μL/min. Fractions containing glycopeptides were purified by in-house packed Stage tips (Empore disk-C18, 3M) and further fractionated by IEF as previously described (35). For each IEF fraction, 50% was analyzed by nLC-MS/MS as described below.…”