Abstract
Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biological regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chemical glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chemical enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chemical labeling, as well as previously unknown protein isoforms including pSPs.
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Acknowledgements
We thank P. Robinson and N. Rumachik for critical reading. Financial support from the US National Institutes of Health (CA200423, C.R.B.), Jane Coffin Childs Memorial Fund (C.M.W.), US National Science Foundation (Graduate Research Fellowship, D.R.S.) and Howard Hughes Medical Institute (C.R.B.) are gratefully acknowledged.
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C.R.B. conceived of and directed the project. C.M.W. designed and synthesized the probe. C.M.W. designed and performed cell culture and enrichment studies. A.T.I. and C.M.W. collected MS data. D.R.S. and C.M.W. optimized the IsoStamp algorithm. C.M.W. analyzed the data and composed the manuscript. K.K.P. performed preliminary studies. All authors revised the manuscript.
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Supplementary Figure 2 Representative western blot analysis of enrichment with probe 1 and Jurkat whole-cell lysate labeled with 100 μM Ac4GalNAz, Ac4ManNAz or DMSO vehicle.
Biotinylated proteins (load) were enriched from the supernatant by affinity-capture with avidin–agarose beads. Avidin–agarose beads were reduced and alkylated, and washed with 1% RapiGest, 6 M urea, and PBS. Beads were checked for anti-biotin signal before and after washing. Treatment with 2% formic acid cleaved probe 1 and released glycoproteins from the agarose beads.
Supplementary Figure 3 Representative western blot analysis of enrichment with probe 1 and MCF7 conditioned medium and lysate labeled with 100 μM Ac4GalNAz, Ac4ManNAz or DMSO vehicle.
A. Protein after tagging by probe 1. B. Protein after enrichment with streptavidin–agarose. C. A sample of the streptavidin–agarose beads after washing (1% RapiGest, 6 M urea, PBS) and reduction/alkylation.
Supplementary Figure 4 Representative western blot analysis of enrichment with probe 1 and PC-3 conditioned medium and lysate labeled with 100 μM Ac4GalNAz, Ac4ManNAz or DMSO vehicle.
A. Protein after tagging by probe 1. B. Protein after enrichment with streptavidin–agarose. C. A sample of the streptavidin–agarose beads after washing (1% RapiGest, 6 M urea, PBS) and reduction/alkylation.
Supplementary Figure 5 IsoStamp-directed glycoproteomics selects isotopically recoded species at a fourfold higher rate across fractions and glycan type from Jurkat cell lysates.
Jurkat cells were labeled with 100 μM Ac4GalNAz or Ac4ManNAz for 48 h. Media (F1), soluble (F2), and insoluble (F3) cellular fractions were enriched for isotopically recoded glycopeptides and analyzed by MS. Tandem MS was collected with an inclusion list (targeted) or by data-dependent analysis of the six most intense ions detected in each full-scan mass spectrum (untargeted).
Supplementary Figure 6 Representative assignments for glycopeptides from MS2 and MS3 spectra for O-GalNAz (a), bis-sialylated O8 (b), and N-glycan N5 (c) glycoforms.
The tagged glycan is denoted with “Br2.” The metabolically labeled glycan (but not tagged) is denoted with “N3.”
Supplementary Figure 7 Medium from Jurkat cells displays core 1 O-glycans.
Medium from Jurkat cells metabolically labeled with 100 μM Ac4GalNAz or DMSO vehicle was treated with neuraminidase and analyzed by staining with FITC–PNA that detects the core 1 O-glycan. Ponceau staining shows equal protein loading (20 μg protein per lane). Lane 1: GalNAz-labeled Jurkat media + neuraminidase. Lane 2: GalNAz-labeled Jurkat media – neuraminidase. Lane 3: DMSO treated Jurkat media + neuraminidase. Lane 4: DMSO treated Jurkat media – neuraminidase.
Supplementary Figure 8 Assigned tandem mass spectra of peptide isoforms identified from Ac4ManNAz-labeled PC-3 cells.
Assigned tandem mass spectra of peptide isoforms identified from Ac4ManNAz-labeled PC-3 cells from (a) prostate-specific microseminoprotein (Q1L6U9), (b) Glucosidase 2 subunit beta (P14314), and (c) Dickkopf-related protein 1 (O94907). Amino acid substitutions are in bold red font. Spectra were assigned with Byonic as a node in Proteome Discoverer.
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Supplementary Figures 1-8, Supplementary Table 1, Supplementary Methods and Supplementary Data 1 (PDF 2596 kb)
Supplementary Data 2
Excel data of all assignments (XLSX 2046 kb)
Supplementary Data 3
Spectra of Jurkat glycopeptide assignments (PDF 55882 kb)
Supplementary Data 4
Spectra of Jurkat glycopeptide assignments (PDF 35042 kb)
Supplementary Data 5
Spectra of PC-3 glycopeptide assignments (PDF 12943 kb)
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Woo, C., Iavarone, A., Spiciarich, D. et al. Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat Methods 12, 561–567 (2015). https://doi.org/10.1038/nmeth.3366
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DOI: https://doi.org/10.1038/nmeth.3366
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