Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry

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 Nglycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and 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 non-redundant 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 (~200 pg/mL), cathepsin L (~1 ng/mL), and transforming growth factor beta 1 (~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.

Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis

“…Although identification of 303 glycoproteins and 639 Nglycosylation sites from human plasma, representing the largest number of glycoproteome identification that has been reported, 36 800 μL of plasma was used in that case. Also extensive sample preparation including depletion of six highabundance proteins and fractionation of the plasma sample into 30 SCX fractions were performed.…”

The D173G mutation in ADAMTS-13 causes a severe form of congenital thrombotic thrombocytopenic purpura

“…ADAMTS-13 undergoes extensive maturation in endoplasmic reticulum (ER) and is heavily glycosylated (44). In particular, the O-fucosylation occurring at 6 TSP1 repeats of ADAMTS13 and the N-glycosylation process appear to be critical for folding, secretion and proteolytic activity of ADAMTS-13 (45).…”

N-glycoprotein profiling of lung adenocarcinoma pleural effusions by shotgun proteomics

“…[4][5][6] This protocol removes the major plasma protein albumin, which is not N-glycosylated, and increases complexity of mass spectrometric analyses. Other high abundance N-GPs in the g/L concentration range are purified.…”