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. 2017 Apr 7;16(4):1706-1718.
doi: 10.1021/acs.jproteome.6b01053. Epub 2017 Feb 28.

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes

Christina M Woo  1   2   3   4 , Alejandra Felix  1   2   3   4 , William E Byrd  1   2   3   4 , Devon K Zuegel  1   2   3   4 , Mayumi Ishihara  1   2   3   4 , Parastoo Azadi  1   2   3   4 , Anthony T Iavarone  1   2   3   4 , Sharon J Pitteri  1   2   3   4 , Carolyn R Bertozzi  1   2   3   4
Affiliations

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes

Christina M Woo et al. J Proteome Res. .

Abstract

Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by IsoTaG were found to be comparable to released N-glycans identified by permethylation analysis. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.

Keywords: IsoStamp; LC−MS/MS; chemical biology; chemical enrichment; glycoconjugate; glycomics; glycoprotein; glycoproteomics; metabolic labeling.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic of IsoTaG method. Cell lines derived from various human tissues were treated with Ac4ManNAz or Ac4GalNAz to generate metabolically labeled glycoproteins. The glycoproteins were tagged with IsoTaG probe mixture 3 and affinity enriched. Tryptic digestion and cleavage of probe mixture 3 recovered isotopically recoded glycopeptides for analysis. Glycopeptides were analyzed by targeted LC–MS using IsoStamp v2.0 to direct selection of isotopically recoded species. A survey of 15 human cell lines was performed to demonstrate the broad applicability of IsoTaG and revealed 74 glycan structures over 3500 intact glycopeptides.
Figure 2
Figure 2
Workflow for model generation and pattern matching by IsoStamp v2.0. (A) Full scan MS data are bundled across scans and peaks are selected for analysis by charge state. (B) A predicted distribution is generated by convolution of a base pattern derived from a modified averagine model and an overlaid tag pattern. (C) Observed and predicted distributions are compared by subtraction to obtain linear and MSQR errors. Examples of errors used in the error function are shown in red overlaid on the observed pattern match shown in gray, where a denotes the abundance of the peak contributing to the error measurement. Error types: m = misaligned, pr = precursor, N = noise, d = m/z deviation, M = missing.
Figure 3
Figure 3
Fidelity of pattern recognition by IsoStamp. (A) Model system design. Bovine serum albumin (BSA) was tagged with halogen tags, digested, and spiked into Jurkat lysates. (B) Structures of halogen tags include tag 1 (monobromide), tag 2 (dichloride), and tag 3 (dibromide). (C) Comparison of false negative rates between IsoStamp v1 and IsoStamp v2 across tags 1–3. Bovine serum albumin tagged with a halogenated arene (tags 1–3) was added to Jurkat whole cell lysate and analyzed by MS. Data were obtained from ref . (D) Score distribution of pattern matches produced by IsoStamp v2 searching for halogen tagged peptides in complex mixtures. Jurkat lysates with halogen tagged BSA peptides generated better scored pattern matches than Jurkat lysate alone. Higher score indicates better match.
Figure 4
Figure 4
Evaluation of stable isotope probes and pattern diversity. (A) Stable isotope probe mixtures 1–8 were prepared by NMR titration and validated by MS. (B) Manual validation of the top 50 scored pattern matches by IsoStamp v2.0 produced by the stable isotope mixtures 1–8. BSA was chemically modified on its lysine residues with azidoacetate, conjugated with the stable isotope mixtures by CuAAC, and analyzed on an Orbitrap XL.
Figure 5
Figure 5
N-Glycan structures observed in a 15-cell line survey. (A) N-Glycans ranged from high-mannose and partially degraded, hybrid, biantennary, triantennary, and tetrantennary glycans. (B) Frequency of observed metabolically labeled N-glycan structures. Bars are color coded to class of glycan structure.
Figure 6
Figure 6
O-Glycan structures observed in a 15-cell line survey. (A) A total of 18 O-glycan structures were observed, including O-GlcNAc (O1, O1*), mucin type (O2–O18), and sialylated glycan structures (e.g., O5–O7). (B) Frequency of observed metabolically labeled O-glycan structures.
Figure 7
Figure 7
Glycoproteins and glycopeptides identified across 15 cell lines. (A) Distribution of glycoprotein subcellular localization. (B) Distribution of glycans labeled by Ac4GalNAz. (C) Distribution of glycan attachment sites labeled by Ac4ManNAz. (D) Frequency of unique peptide substrate occurrence across cell lines. The majority of peptide substrates were observed in only one cell line.
Figure 8
Figure 8
Comparison of N-glycan structures by released N-glycan analysis and glycans observed by IsoTaG from a single cell line. (A) Released permethylated N-glycan analysis by MALDI. Glycan structures from PC-3 cells also observed by IsoTaG are marked with “*”. (B) Intact glycan analysis by IsoTaG of PC-3 cells. Glycan structures from PC-3 cells that are also found by free glycan analysis are shown in black. Structure codes correspond to the glycan structures in Figure 5.
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