title: 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 O-GalNAc attached to tyrosine. Furthermore, we uncovered ≈ 220 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.
authors: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. seva@sund.ku.dk
authors: Sergey Y
authors: SY
authors: Vakhrushev
authors: Catharina
authors: C
authors: Steentoft
authors: Malene B
authors: MB
authors: Vester-Christensen
authors: Eric P
authors: EP
authors: Bennett
authors: Henrik
authors: H
authors: Clausen
authors: Steven B
authors: SB
authors: Levery
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D002918:Chymotrypsin / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D010446:Peptide Fragments; D020543:Proteome; D014443:Tyrosine; D002918:Chymotrypsin; D014357:Trypsin; D000116:Acetylgalactosamine
references: Nat Rev Mol Cell Biol. 2003 Apr;4(4):295-308
references: 12671652
references: J Biol Chem. 2010 Dec 17;285(51):40097-103
references: 20952385
references: Trends Cell Biol. 2011 Mar;21(3):149-58
references: 21145746
references: Glycobiology. 2012 Jun;22(6):736-56
references: 22183981
references: Eur J Mass Spectrom (Chichester). 2010;16(3):421-8
references: 20530845
references: Mol Cell Proteomics. 2012 Aug;11(8):215-29
references: 22645316
references: Proteomics. 2008 Dec;8(23-24):4862-72
references: 19003865
references: Nat Methods. 2009 Nov;6(11):809-11
references: 19838169
references: Mol Cell Proteomics. 2007 Jan;6(1):141-9
references: 17074749
references: Proc Natl Acad Sci U S A. 2012 Jun 19;109(25):9893-8
references: 22566642
references: Annu Rev Biochem. 2011;80:825-58
references: 21391816
references: Nat Biotechnol. 2003 Jun;21(6):660-6
references: 12754519
references: Nat Methods. 2011 Oct 09;8(11):977-82
references: 21983924
references: J Biol Chem. 2010 Nov 19;285(47):36293-303
references: 20837471
references: Mol Cell Proteomics. 2007 Oct;6(10):1778-87
references: 17623646
references: Mol Cell Neurosci. 2002 Apr;19(4):539-51
references: 11988021
references: Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):11848-53
references: 21712440
references: J Cell Biol. 2001 Jun 11;153(6):1277-86
references: 11402070
references: J Proteome Res. 2006 Jul;5(7):1721-30
references: 16823980
references: Mol Cell Neurosci. 2008 Nov;39(3):452-64
references: 18775496
references: J Proteome Res. 2003 Jan-Feb;2(1):59-67
references: 12643544
references: J Proteome Res. 2010 Mar 5;9(3):1323-9
references: 20113005
references: Blood. 2000 Oct 15;96(8):2765-74
references: 11023510
references: J Biol Chem. 2006 Jul 7;281(27):18370-7
references: 16638743
references: Mol Cell Proteomics. 2009 Nov;8(11):2515-26
references: 19674964
references: Annu Rev Biochem. 2003;72:643-91
references: 12676797
references: Mol Cell Proteomics. 2012 Jul;11(7):O111.016774
references: 22393263
references: Mol Cell Proteomics. 2012 Apr;11(4):M111.013649
references: 22171320
references: J Biol Chem. 1998 Oct 16;273(42):27749-54
references: 9765313
references: Glycobiology. 2005 Feb;15(2):153-64
references: 15385431
references: Proteomics. 2007 Aug;7(15):2522-9
references: 17610206
references: J Cell Biol. 2010 May 31;189(5):843-58
references: 20498016
references: Glycobiology. 2012 Jul;22(7):918-29
references: 22422444
references: Anal Bioanal Chem. 2010 Aug;397(8):3421-30
references: 20517600
references: Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13841-6
references: 11095749
references: J Proteome Res. 2009 Feb;8(2):631-42
references: 19093876
references: J Biol Chem. 2011 Nov 18;286(46):40122-32
references: 21937429
references: Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8894-9
references: 19458039
references: J Bone Miner Res. 2007 Feb;22(2):235-42
references: 17129170