Site-specific protein glycosylation analysis with glycan isomer differentiation (original) (raw)

Abstract

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single _N_-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five _N_-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one _N_-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five _O_-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

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Overlaid chromatograms and associated structural assignments of glycopeptides from bovine κ-casein. Color denotes the site(s) of glycosylation from which the glycopeptide originated

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Abbreviations

bLF:

Bovine lactoferrin

CID:

Collision-induced dissociation

CNBr:

Cyanogen bromide

ECC:

Extracted compound chromatogram

Fuc:

Fucose

Gal:

Galactose

Glc:

Glucose

Hex:

Hexose

HexNAc:

_N_-acetylhexosamine

IgG:

Immunoglobulin G

Man:

Mannose

Man_X_ (where X = 5 to 9):

High mannose glycan of composition GlcNAc2Man X

NeuAc:

_N_-acetylneuraminic acid

PGC:

Porous graphitized carbon

Q-TOF:

Quadrupole time-of-flight

RNAse B:

Ribonuclease B

TIC:

Total ion chromatogram

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Acknowledgments

We would like to thank Ning Tang and Keith Waddell (Agilent Technologies Inc.) for instrumentation and technical support. Financial support was provided by the University of California Discovery Grant Program, the California Dairy Research Foundation, the NIEHS Superfund Research Program (P42 ES02710), and the CHARGE Study (P01 ES11269).

Author information

Authors and Affiliations

  1. Department of Chemistry, University of California, Davis, CA, 95616, USA
    Serenus Hua, Charles C. Nwosu, John S. Strum, Richard R. Seipert & Carlito B. Lebrilla
  2. Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764, South Korea
    Hyun Joo An
  3. Department of Food Science and Technology, University of California, Davis, CA, 95616, USA
    Angela M. Zivkovic & J. Bruce German
  4. Foods for Health Institute, University of California, Davis, CA, 95616, USA
    Angela M. Zivkovic, J. Bruce German & Carlito B. Lebrilla
  5. Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, 95616, USA
    Carlito B. Lebrilla

Authors

  1. Serenus Hua
  2. Charles C. Nwosu
  3. John S. Strum
  4. Richard R. Seipert
  5. Hyun Joo An
  6. Angela M. Zivkovic
  7. J. Bruce German
  8. Carlito B. Lebrilla

Corresponding author

Correspondence toCarlito B. Lebrilla.

Additional information

Published in the special issue High-Resolution Mass Spectrometry with guest editors Hans H. Maurer and David C. Muddiman.

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Hua, S., Nwosu, C.C., Strum, J.S. et al. Site-specific protein glycosylation analysis with glycan isomer differentiation.Anal Bioanal Chem 403, 1291–1302 (2012). https://doi.org/10.1007/s00216-011-5109-x

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