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. 2017 Jan;409(2):579-588.
doi: 10.1007/s00216-016-9934-9. Epub 2016 Sep 30.

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

Christina M Woo  1 , Alejandra Felix  1 , Lichao Zhang  2 , Joshua E Elias  2 , Carolyn R Bertozzi  3   4
Affiliations

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

Christina M Woo et al. Anal Bioanal Chem. 2017 Jan.

Abstract

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

Keywords: Chemical proteomics; Glycoproteomics; LC-MS/MS; Metabolic labeling; Sialic acid.

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Conflict of interest statement

Conflict of Interest: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical probe design and application in IsoTaG. A. Complementary probe and glycan pairs. Silane probe 1 reacts selectively with azido sugars (e.g., Ac4ManNAz) glycoproteins. Silane probe 2 reacts selectively with alkynyl sugars (e.g., Ac4ManNAl) glycoproteins. B. Application of IsoTaG to sialylated glycans expressed on the cell surface and secreted.
Figure 2
Figure 2
Global analysis of glycoproteins and glycopeptides identified by IsoTaG analysis of conditioned media from metabolically labeled PC-3 cells. A. Anti-biotin Western blot analysis of tagging and enrichment efficiency with DMSO control, Ac4ManNAl, and Ac4ManNAz samples. Total protein levels shown alongside (ponceau). Lanes: (1) protein input following CuAAC reaction with probe 1 or 2, (2) protein supernatant following overnight incubation with streptavidin–agarose beads, (3) aliquot of streptavidin–agarose beads (10 µL) after enrichment. B. Venn diagram of glycoproteins identified from nonconjugated on-bead tryptic peptides from Ac4ManNAz and Ac4ManNAl samples. C. Subcellular localization of proteins identified by direct intact glycopeptide assignments.
Figure 3
Figure 3
Intact sialylated glycan structures and frequency of occurrence. A. Intact sialylated glycans identified (S1–S8). S1–S3 are O-glycans. S4–S8 are N-glycans. B. Frequency of sialyl glycans observed by spectral counting.
Figure 4
Figure 4
Example spectral assignment of the glycopeptide YSQAVPAVTEGPIPEVLK from cathepsin D carrying SiaNAl (A) or SiaNAz (B) incorporation to glycan S2. Assignment of glycan and peptide fragments enables localization of sialic acid in the glycan structure. The relative abundance of highlighted peaks is reported in ESM Tables S1 and S2.
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