Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry

J Proteome Res. Nov-Dec 2005;4(6):2070-80. doi: 10.1021/pr0502065.

Abstract

The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Blood Proteins / chemistry*
  • Cations
  • Chromatography, Ion Exchange
  • Chromatography, Liquid
  • Glycopeptides / chemistry
  • Glycoproteins / chemistry*
  • Glycosylation
  • Humans
  • Hydrazines / chemistry*
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / chemistry
  • Peptides / chemistry
  • Proteins / chemistry
  • Proteome
  • Proteomics / methods*
  • Spectroscopy, Fourier Transform Infrared
  • Subcellular Fractions

Substances

  • Blood Proteins
  • Cations
  • Glycopeptides
  • Glycoproteins
  • Hydrazines
  • Peptides
  • Proteins
  • Proteome
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase