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, 4 (6), 2070-80

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

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

Tao Liu et al. J Proteome Res.

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.

Figures

Figure 1
Figure 1
Strategy for plasma N-glycoprotein analysis using immunoaffinity subtraction, glycoprotein capture, and mass spectrometry. Crude plasma (red balls marked with “H” represent high abundance proteins; blue balls marked with “L” represent low abundance proteins) is first subjected to multi-component depletion using the pre-packed MARS column, followed by incubating the flow-through (minus the retained abundant proteins) with hydrazide resin. The captured glycoproteins (blue balls marked with “G”) are then washed and digested on-resin with trypsin, and the N-glycopeptides are specifically released from the resin by PNGase F. The resulting deglycosylated peptides can either be identified by 2-D LC-MS/MS or directly analyzed by LC-FTICR to validate the number of glycosylation sites.
Figure 2
Figure 2
SCX fractionation and LC-MS/MS analysis. (A) The deglycosylated peptides were separated into 30 fractions using SCX chromatography. (B) Base peak chromatogram of the LC-MS/MS analysis for fraction 14 (marked with arrow in A). (C) MS spectrum for the peptides eluted at 39.41 min in the base peak chromatogram. (D) MS spectrum for the peptides eluted at 44.83 min in the same base peak chromatogram. (E) Fragmentation of low-abundance ion with m/z=638.3 (marked with asterisk in C) resulted in identification of the fully tryptic peptide K.YSVAN*DTGFVDIPKQEK.A (asterisk indicates the N-glycosylation site and the consensus motif is in bold) and originates from the low-abundance protein cathepsin L. Fragment ions that matched predicted ions are labeled as b and y ions. (F) This peptide was identified from the fragmentation of low-abundance ion with m/z=923.0 (marked with asterisk in D) as the fully tryptic peptide R.LQLEAVN*ITDLSENRK.Q and originated from the low-abundance protein interleukin-1 receptor antagonist protein.
Figure 3
Figure 3
Overlap between the identified proteins from the specifically enriched N-glycopeptides and a previous study based on global tryptic peptides (Ref 30). In the previous study, the global tryptic peptides were prepared by digesting the plasma proteins with and without albumin depletion or IgG depletion, fractionated by SCX chromatography, and analyzed by LC-MS/MS with and without enhanced separation (e.g., longer gradient, smaller i.d. column), which resulted in a total of 938 non-redundant protein identifications. In contrast, 303 non-redundant N-glycoproteins were confidently identified from 30 regular LC-MS/MS analyses of the deglycosylated peptides prepared by using MARS depletion, glycoprotein capture, and SCX fractionation.
Figure 4
Figure 4
Comparison of protein categorization using GO component terms. Major component categories are shown for N-glycoprotein identifications (A) and global protein identifications (B).

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