Human Plasma <i>N</i>-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry

TLDR

The human plasma proteome is highly complex, spanning over ten orders of magnitude in protein abundance and exhibiting extensive heterogeneity from post‑translational modifications such as glycosylation, which hampers current analytical methods. This study presents a strategy combining immunoaffinity subtraction and glycoprotein capture to enable comprehensive analysis of plasma N‑glycoproteins by reducing protein concentration range and sample complexity. Six high‑abundance proteins were removed with an immobilized antibody column, followed by hydrazide‑based capture of N‑linked glycoproteins, enzymatic digestion, PNGase F‑mediated release of glycopeptides, SCX fractionation, and LC‑MS/MS analysis of the deglycosylated peptides. The workflow identified 2,053 distinct N‑glycopeptides corresponding to 303 non‑redundant glycoproteins, including low‑abundance targets such as interleukin‑1 receptor antagonist, cathepsin L, and TGF‑β1, and yielded 639 glycosylation sites with high accuracy confirmed by LC‑FTICR mass spectrometry. Keywords: human plasma, mass spectrometry, proteomics, N‑glycosylation, immunoaffinity subtraction.

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 (∼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. Keywords: human plasma • mass spectrometry • proteomics • N-glycosylation • immunoaffinity subtraction