Supplementary Table 1. Proteins selectively identified with high confidence in GlcNAlk samples by mass spectrometry.Data was considered high confidence if the number of assigned spectra was at least 10 fold greater for GlcNAlk samples than DMSO control samples. Further, the protein must have been identified with spectral counts greater than or equal to 5. Data was compared to proteomic data sets from Wells et al (1), Teo et al (2), Gurcel et al (3), Cieniewski-Bernard et al (4), Clark et al (5), Sprung et al (6), Nandi et al (7), Wang et al (8), Vosseller et al (9), Khidekel et al 2007 (10), Hanover et al (11), Khidekel et al 2004 (12), Dehennaut et al (13), and Boyce et al (14). and proteins were shaded blue if they have not been previously identified. Nuclear pore glycoprotein p62, Lamin-A/C, HP1γ, and NEDD4-1 (confirmed by Western blotting) are highlighted in yellow.
Identified Proteins (142, 79 new) / Accession Number / GlcNAlk / DMSO / Citation / FunctionGlyceraldehyde-3-phosphate dehydrogenase / IPI00273646 (+2) / 447 / 15 / Wells, Teo, Gurcel, Cienewski-Bernard, Dehennaut, Wang / Metabolism
Cofilin-1 / IPI00890117 / 131 / 0 / Wells, Teo, Nandi / Structural
Triosephosphate isomerase / IPI00467833 / 129 / 8 / Teo, Cieniewski-Bernard, Sprung, Clark / Metabolism
Annexin A2 / IPI00468203 / 127 / 3 / Clark, Gurcel / Structural
Elongation factor 2 / IPI00466069 / 124 / 0 / Clark, Teo / Protein Synthesis, Quality Control, & Turnover
Isoform A of Lamin-A/C / IPI00620256 / 67 / 3 / Structural
Putative uncharacterized protein / IPI00229080 / 62 / 6 / Unknown
S-methyl-5'-thioadenosine phosphorylase / IPI00132096 / 62 / 0 / Nandi / Metabolism
Annexin A1 / IPI00230395 / 59 / 3 / Wells, Teo, Nandi / Signaling
E3 ubiquitin-protein ligase NEDD4 / IPI00462445 / 59 / 0 / Protein Synthesis, Quality Control, & Turnover
Vimentin / IPI00227299 / 59 / 3 / Wang, Teo / Structural
L-lactate dehydrogenase A chain / IPI00319994 (+1) / 58 / 0 / Dehennaut, Wang, / Metabolism
Isoform M1 of Pyruvate kinase isozymes M1/M2 / IPI00407130 / 57 / 0 / Clark, Teo, Gurcel, Sprung, Wells, Dehennaut, Nandi / Metabolism
Isoform 2 of Tropomyosin alpha-3 chain / IPI00230044 / 54 / 0 / Structural
Heterogeneous nuclear ribonucleoprotein F / IPI00226073 / 43 / 0 / RNA Processing
Voltage-dependent anion-selective channel protein 3 / IPI00876341 / 41 / 0 / Intracellular Trafficking
Destrin / IPI00127942 / 40 / 0 / Structural
14-3-3 protein epsilon / IPI00118384 / 34 / 2 / Sprung, Clark / Signaling
Isoform 1 of Heterogeneous nuclear ribonucleoprotein K / IPI00223253 (+3) / 34 / 0 / Wang, Clark, Nandi, Gurcel / RNA Processing
Voltage-dependent anion-selective channel protein 2 / IPI00122547 / 31 / 0 / Clark / Intracellular Trafficking
Isoform 1 of Heterogeneous nuclear ribonucleoprotein A3 / IPI00269661 (+1) / 30 / 0 / Clark, Teo, Wang, Nandi / RNA Processing
Poly(rC)-binding protein 1 / IPI00128904 / 28 / 0 / Nandi / Protein Synthesis, Quality Control, & Turnover
Zyxin / IPI00387422 (+1) / 28 / 0 / Structural
Chromobox protein homolog 3, HP1γ / IPI00129468 (+1) / 27 / 0 / Gene Regulation & Transcription
Nucleolar protein 58 / IPI00463468 / 27 / 0 / Protein Synthesis, Quality Control, & Turnover
Host cell factor C1 / IPI00828490 (+1) / 26 / 0 / Wells, Wang, Teo, Vosseller, Khidekel (2007), Khidekel (2004), / Gene Regulation & Transcription
Isoform 1 of 14-3-3 protein theta / IPI00408378 (+2) / 25 / 0 / Gurcel, Clark / Signaling
AHNAK nucleoprotein isoform 1 / IPI00553798 / 22 / 0 / Signaling
caldesmon 1 / IPI00122450 (+4) / 22 / 0 / Structural
Eukaryotic translation initiation factor 5A-1 / IPI00108125 (+1) / 22 / 0 / Nandi / Protein Synthesis, Quality Control, & Turnover
Adenylyl cyclase-associated protein 1 / IPI00137331 (+1) / 20 / 0 / Structural
Alpha-actinin-1 / IPI00380436 / 20 / 0 / Nandi / Structural
Asparagine synthetase [glutamine-hydrolyzing] / IPI00116966 / 20 / 0 / Metabolism
Bifunctional aminoacyl-tRNA synthetase / IPI00339916 / 20 / 0 / RNA Processing
Isoform 1 of Filamin-A / IPI00131138 (+3) / 20 / 0 / Nandi, Gurcel / Structural
MICAL-like 2 / IPI00280103 (+1) / 20 / 0 / Structural
Myosin-9 / IPI00123181 / 20 / 0 / Nandi, Teo, Cienewski-Bernard / Structural
Protein S100-A4 / IPI00124096 / 20 / 0 / Signaling
Thioredoxin / IPI00226993 / 20 / 0 / Teo / Signaling
Isoform 2 of F-actin-capping protein subunit beta / IPI00269481 (+2) / 19 / 0 / Clark / Structural
caprin-1 isoform c / IPI00121515 (+2) / 18 / 0 / Teo / Protein Synthesis, Quality Control, & Turnover
Cathepsin B / IPI00113517 / 18 / 0 / Signaling
Isoform Long of Delta-1-pyrroline-5-carboxylate synthetase / IPI00129350 (+2) / 16 / 0 / Wang / Metabolism
T-complex protein 1 subunit beta / IPI00320217 / 15 / 0 / Clark / Protein Synthesis, Quality Control, & Turnover
Tubulin beta-5 chain / IPI00117352 / 15 / 4 / Clark, Wang, Teo, Sprung, Gurcel / Structural
Cadherin-3 / IPI00109340 (+2) / 14 / 0 / Signaling
Far upstream element-binding protein 2 / IPI00462934 / 14 / 0 / Nandi / RNA Processing
Eukaryotic translation initiation factor 4B / IPI00221581 / 13 / 0 / Protein Synthesis, Quality Control, & Turnover
Myb-binding protein 1A / IPI00331361 / 13 / 0 / Gene Regulation & Transcription
Putative uncharacterized protein / IPI00132089 / 13 / 0 / Unknown
similar to high-mobility group box 1 / IPI00665601 / 13 / 0 / Unknown
similar to Nucleophosmin / IPI00849626 / 13 / 0 / Unknown
ADP/ATP translocase 2 / IPI00127841 / 12 / 0 / Clark, Teo, Wang / Metabolism
Elongation factor 1-gamma / IPI00318841 / 12 / 0 / Protein Synthesis, Quality Control, & Turnover
Glycyl-tRNA synthetase / IPI00112555 / 12 / 0 / Nandi / RNA Processing
Heat shock protein HSP 90-alpha / IPI00330804 / 12 / 0 / Nandi, Teo, Wells / Protein Synthesis, Quality Control, & Turnover
Inosine-5'-monophosphate dehydrogenase 2 / IPI00323971 (+1) / 12 / 0 / Nandi, Teo / Metabolism
Isoform 1 of Protein arginine N-methyltransferase 1 / IPI00120495 (+3) / 12 / 0 / Signaling
LIM domain and actin-binding protein 1 isoform a / IPI00112339 / 12 / 0 / Structural
Peroxiredoxin-1 / IPI00121788 (+2) / 12 / 0 / Metabolism
CTP synthase 1 / IPI00111959 / 11 / 0 / Metabolism
deoxyuridine triphosphatase isoform 1 / IPI00187434 (+1) / 11 / 0 / Metabolism
Glutathione S-transferase omega-1 / IPI00114285 / 11 / 0 / Wang, Sprung / Protein Synthesis, Quality Control, & Turnover
Homeobox protein engrailed-2 / IPI00020031 / 11 / 0 / Gene Regulation & Transcription
Isoform 1 of Nuclear autoantigenic sperm protein / IPI00130959 (+1) / 11 / 2 / Gene Regulation & Transcription
Isoform 5 of Ubiquitin-associated protein 2-like / IPI00407835 (+1) / 11 / 0 / Clark, Teo / Signaling
Nucleolar RNA helicase 2 / IPI00652987 / 11 / 0 / RNA Processing
Src substrate cortactin / IPI00118143 (+1) / 11 / 0 / Teo / Signaling
Cytochrome P450 4V3 / IPI00120197 / 10 / 0 / Metabolism
Isoform 2 of Fatty aldehyde dehydrogenase / IPI00394758 / 10 / 0 / Metabolism
Isoform CW17 of Splicing factor 1 / IPI00116284 (+4) / 10 / 0 / Teo / Gene Regulation & Transcription
Uncharacterized protein / IPI00830960 / 10 / 0 / Unknown
Actin, alpha skeletal muscle / IPI00110827 (+4) / 9 / 0 / Clark / Structural
Calponin-2 / IPI00116649 / 9 / 0 / Signaling
Elongation factor 1-alpha 1 / IPI00307837 / 9 / 0 / Nandi, Teo, Wang, Gurcel, Wells, Clark / Protein Synthesis, Quality Control, & Turnover
Isoform 1 of Lipoma-preferred partner homolog / IPI00221494 / 9 / 0 / Signaling
Isoform 1 of Poly(rC)-binding protein 2 / IPI00127707 (+1) / 9 / 0 / Clark, Gurcel / Protein Synthesis, Quality Control, & Turnover
Isoform 3 of A-kinase anchor protein 2 / IPI00336504 (+5) / 9 / 0 / RNA Processing
PDZ and LIM domain protein 5 isoform ENH1 / IPI00653381 (+1) / 9 / 0 / Structural
Putative uncharacterized protein / IPI00136883 (+1) / 9 / 0 / Unknown
Putative uncharacterized protein / IPI00653643 / 9 / 0 / Unknown
Tropomyosin alpha-4 chain / IPI00421223 / 9 / 0 / Structural
40S ribosomal protein S12 / IPI00225634 (+2) / 8 / 0 / Protein Synthesis, Quality Control, & Turnover
D-3-phosphoglycerate dehydrogenase / IPI00225961 / 8 / 0 / Nandi, Clark / Metabolism
Ewing sarcoma breakpoint region 1 / IPI00322492 (+3) / 8 / 0 / Wells, Teo / Unknown
Histone-binding protein RBBP7 / IPI00122698 (+1) / 8 / 0 / Gene Regulation & Transcription
Isoform 1 of Plasminogen activator inhibitor 1 RNA-binding protein / IPI00471475 (+3) / 8 / 0 / Teo / RNA Processing
Isoform Smooth muscle of Myosin light polypeptide 6 / IPI00354819 (+3) / 8 / 0 / Nandi / Structural
Matrin-3 / IPI00453826 / 8 / 0 / Gene Regulation & Transcription
Peroxiredoxin-4 / IPI00116254 / 8 / 0 / Nandi / Metabolism
similar to LOC446231 protein / IPI00116718 (+1) / 8 / 0 / unknown
Vesicle-associated membrane protein-associated protein A / IPI00125267 / 8 / 0 / Intracellular Trafficking
Cytochrome b5 type B / IPI00315794 / 7 / 0 / Metabolism
Impa1 protein / IPI00473176 (+1) / 7 / 0 / Metabolism
Isoform 1 of Glyoxalase domain-containing protein 4 / IPI00110721 (+1) / 7 / 0 / Metabolism
Multifunctional protein ADE2 / IPI00322096 (+1) / 7 / 0 / Teo / Metabolism
Protein PRRC1 / IPI00130462 / 7 / 0 / Teo / Unknown
Putative pre-mRNA-splicing factor ATP-dependent RNA Processing helicase DHX15 / IPI00128818 / 7 / 0 / RNA Processing
Putative uncharacterized protein / IPI00126248 / 7 / 0 / Unknown
Transmembrane protein 117 / IPI00221687 / 7 / 0 / Signaling
Xanthine dehydrogenase/oxidase / IPI00352984 (+1) / 7 / 0 / Metabolism
40S ribosomal protein S3 / IPI00134599 / 6 / 0 / Dehennaut, Clark, Wang / Protein Synthesis, Quality Control, & Turnover
Anamorsin / IPI00187301 / 6 / 0 / Signaling
ATP-binding cassette sub-family E member 1 / IPI00322869 / 6 / 0 / Intracellular Trafficking
Calreticulin / IPI00123639 / 6 / 0 / Nandi, Sprung / Protein Synthesis, Quality Control, & Turnover
eukaryotic peptide chain release factor GTP-binding subunit ERF3A isoform 1 / IPI00230355 (+1) / 6 / 0 / Protein Synthesis, Quality Control, & Turnover
High mobility group protein HMGI-C / IPI00331612 / 6 / 0 / Gene Regulation & Transcription
Isoform 1 of Integrin beta-like protein 1 / IPI00123829 / 6 / 0 / Signaling
Isoform 1 of La-related protein 1 / IPI00929786 / 6 / 0 / RNA Processing
Isoform 3 of Ribosome-binding protein 1 / IPI00121149 (+1) / 6 / 0 / Intracellular Trafficking
SUMO-activating enzyme subunit 2 / IPI00023234 / 6 / 0 / Protein Synthesis, Quality Control, & Turnover
Transgelin-2 / IPI00125778 / 6 / 0 / Nandi / Structural
Tubby-related protein 1 / IPI00130590 (+3) / 6 / 0 / Signaling
Ubiquitin thioesterase OTUB1 / IPI00154004 (+1) / 6 / 0 / Clark / Protein Synthesis, Quality Control, & Turnover
WW domain-binding protein 11 / IPI00123333 / 6 / 0 / RNA Processing
182 kDa tankyrase-1-binding protein / IPI00459443 / 5 / 0 / Signaling
60S acidic ribosomal protein P0 / IPI00314950 / 5 / 0 / Teo, Wang / Protein Synthesis, Quality Control, & Turnover
60S ribosomal protein L10a / IPI00127085 (+1) / 5 / 0 / Teo / Protein Synthesis, Quality Control, & Turnover
Aldose reductase-related protein 2 / IPI00273096 / 5 / 0 / Metabolism
BAT2 domain-containing protein 1 / IPI00330171 (+2) / 5 / 0 / Wang / Unknown
Branched-chain-amino-acid aminotransferase / IPI00653423 (+3) / 5 / 0 / Metabolism
BRI3-binding protein / IPI00226771 / 5 / 0 / Signaling
Calcyclin-binding protein / IPI00115650 / 5 / 0 / Protein Synthesis, Quality Control, & Turnover
Crk-like protein / IPI00113362 / 5 / 0 / Signaling
Elongation factor 1-beta / IPI00320208 / 5 / 0 / Sprung / Protein Synthesis, Quality Control, & Turnover
Four and a half LIM domains 3 / IPI00828338 / 5 / 0 / Structural
Gamma-sarcoglycan / IPI00110503 / 5 / 0 / Structural
Glutamate--cysteine ligase regulatory subunit / IPI00114329 / 5 / 0 / Metabolism
Histone H1.2 / IPI00223713 / 5 / 0 / Teo / Gene Regulation & Transcription
Isoform 1 of Eukaryotic translation initiation factor 4 gamma 1 / IPI00421179 (+3) / 5 / 0 / Nandi / Protein Synthesis, Quality Control, & Turnover
Isoform Alpha of Lamina-associated polypeptide 2, isoforms alpha/zeta / IPI00126338 (+1) / 5 / 0 / Gene Regulation & Transcription
KH domain-containing, RNA-binding, signal transduction-associated protein 1 / IPI00458765 / 5 / 0 / RNA Processing
neurogenic locus notch homolog protein 2 / IPI00467908 / 5 / 0 / Signaling
Nuclear pore glycoprotein p62 / IPI00139994 / 5 / 0 / Hanover, Teo, Wells / Intracellular Trafficking
Nucleolin / IPI00317794 / 5 / 0 / Teo, Wang / Gene Regulation & Transcription
Nucleoplasmin-3 / IPI00131725 / 5 / 0 / Teo / RNA Processing
olfactory receptor 585 / IPI00127945 (+2) / 5 / 0 / Signaling
Peptidyl-prolyl cis-trans isomerase FKBP4 / IPI00230139 / 5 / 0 / Clark, Teo / Protein Synthesis, Quality Control, & Turnover
Ran-specific GTPase-activating protein (Fragment) / IPI00321978 / 5 / 0 / Teo / Signaling
Ribonucleoside-diphosphate reductase large subunit / IPI00315127 (+1) / 5 / 0 / Nandi / Metabolism
Ubiquitin-40S ribosomal protein S27a / IPI00470152 / 5 / 0 / Protein Synthesis, Quality Control, & Turnover
Isoform 1 of Nesprin-2 / IPI00845851 / 5 / 0 / Structural
1.Wells L, Vosseller K, Cole RN, Cronshaw JM, Matunis MJ, Hart GW. (2002) Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1:791-804.
2.Teo CF, et al. (2010) Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6:338-343.
3.Gurcel C, et al. Identification of new O-GlcNAc modified proteins using click-chemistry-based tagging. Anal Bioanal Chem 390:2089-2097.
4.Cieniewski-Bernard C, et al. (2004) Identification of O-linked N-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry. Mol Cell Proteomics 3:577-585.
5.Clark PM, et al. (2008) Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J Am Chem Soc 130:11576-11577.
6.Sprung R, et al. (2005) Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J Proteome Res. 4:950-957.
7.Nandi A, et al. (2006) Global identification of O-GlcNAc-modified proteins. Anal Chem 78:452-458.
8.Wang Z, Pandey A, Hart GW. (2007) Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation. Mol Cell Proteomics 6:1365-1379.
9.Vosseller K, et al. (2006) O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics 5:923-934.
10.Khidekel N, et al. (2007) Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol 3:339-348.
11.Hanover JA, Cohen CK, Willingham MC, Park MK. (1987) O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J Biol Chem 20:9887-9894.
12.Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. (2004) Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA 101:13132-13137.
13.Dehennaut V, et al. (2008) Identification of Structural and Functional O-linked N-Acetylglucosamine-bearing Proteins in Xenopus laevis Oocyte. Mol Cell Proteomics 7:2229-2245.
14.Boyce M, et al. (2011) Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Nat Acad Sci USA 108:3141-3146.
1