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. 2019 Aug;18(8):1479-1490.
doi: 10.1074/mcp.RA119.001411. Epub 2019 May 16.

Glycoproteogenomics: A Frequent Gene Polymorphism Affects the Glycosylation Pattern of the Human Serum Fetuin/α-2-HS-Glycoprotein

Yu-Hsien Lin  1 , Jing Zhu  1 , Sander Meijer  2 , Vojtech Franc  3 , Albert J R Heck  4
Affiliations

Glycoproteogenomics: A Frequent Gene Polymorphism Affects the Glycosylation Pattern of the Human Serum Fetuin/α-2-HS-Glycoprotein

Yu-Hsien Lin et al. Mol Cell Proteomics. 2019 Aug.

Abstract

Fetuin, also known as α-2-HS-glycoprotein (gene name: AHSG), is one of the more abundant glycoproteins secreted into the bloodstream. There are two frequently occurring alleles of human AHSG, resulting in three genotypes (AHSG*1, AHSG*2, and heterozygous AHSG1/2). The backbone amino acid sequences of fetuin coded by the AHSG*1 and AHSG*2 genes differ in two amino acids including one known O-glycosylation site (aa position 256). Although fetuin levels have been extensively studied, the originating genotype is often ignored in such analysis. As fetuin has been suggested repeatedly as a potential biomarker for several disorders, the question whether the gene polymorphism affects the fetuin profile is of great interest. In this work, we describe detailed proteoform profiles of fetuin, isolated from serum of 10 healthy and 10 septic patient individuals and investigate potential glycoproteogenomics correlations, e.g. how gene polymorphisms affect glycosylation. We established an efficient method for fetuin purification from individuals' serum using ion-exchange chromatography. Subsequently, we performed hybrid mass spectrometric approaches integrating data from native mass spectra and peptide-centric MS analysis. Our data reveal a crucial effect of the gene polymorphism on the glycosylation pattern of fetuin. Moreover, we clearly observed increased fucosylation in the samples derived from the septic patients. Our serum proteoform analysis, targeted at one protein obtained from 20 individuals, exposes the wide variability in proteoform profiles, which should be taken into consideration when using fetuin as biomarker. Importantly, focusing on a single or few proteins, the quantitative proteoform profiles can provide, as shown here, already ample data to classify individuals by genotype and disease state.

Keywords: Glycoproteins*; Glycoproteomics; Plasma or serum analysis; Post-translational modifications*; Protein Identification*; fetuin.

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Figures

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Graphical abstract
Fig. 1.
Fig. 1.
Different genotypes lead to different proteoform profiles. Three representative zero-charge deconvoluted native mass spectra of fetuin purified from serum of A, donor F1 (AHSG*1), B, donor M3 (AHSG*2), and C, donor F3 (AHSG1/2). Corresponding proteoforms between AHSG*1 and AHSG*2 differ in mass from each other by 16 Da (as shown for the proteoforms with the mass of 43,272.88 Da in A and 43,289.26 Da in B). In B, the proteoform with the mass of 42,633.02 Da differs from the most abundant proteoform by 656 Da, which agrees to an O-glycan composition of HexNAc1Hex1Neu5Ac1. This proteoform (42,632.24 Da) can be observed in C, but is fully absent in A.
Fig. 2.
Fig. 2.
Peptide signatures of distinct fetuin genotypes. EThcD MS/MS spectra of the peptide A, 243VAVTCTVFQTQPVTSQPQPE262 (AHSG*1) and B, 243VAVTCMVFQTQPVSSQPQPE262 (AHSG*2), obtained by proteolytic digestion (Trypsin + Glu-C), harboring the two mutation sites. The EThcD spectra provide confirmation of the unique peptide sequence and position of the O-glycan, being Thr256 in AHSG*1. Spectra were acquired for the precursor ions with three charges and m/z of 958.78, and 745.03 for AHSG*1 and AHSG*2, respectively as these peptides were predominantly non-modified for AHSG*2 and fully modified for AHSG*1. C, Quantification of the peptide signatures, containing the mutations and O-glycosylation site Thr/Ser256. In these bars, the abundance of these peptides was averaged over all genotype-specific fetuin samples purified from the serum of the 10 healthy individuals across all three genotypes, AHSG*1 (n = 4), AHSG*2 (n = 2) and AHSG1/2 (n = 4). Relative abundances of peptide proteoforms were estimated from their corresponding extracted ion chromatograms (XICs) and normalized to 100%. The AHSG*1 and AHSG*2 signature peptides contain the Thr256 and Ser256 site, respectively. Both peptides can be extracted and separately quantified for the heterozygote AHSG1/2 donors. Black bar is unmodified, blue bar is modified by HexNAc1Hex1 with one sialic acid and red bar is modified by HexNAc1Hex1 harboring two sialic acids.
Fig. 3.
Fig. 3.
Comprehensive, quantitative and site-specific annotation of fetuin proteoform profiles. The site-specifically annotated zero-charge deconvoluted native mass spectrum of fetuin pooled from human sera of various donors. The overall PTM compositions of the most abundant proteoforms are color coded. Each color represents a glycan composition without the sialic acids; the number of sialic acids attached is marked on the top of each peak. All displayed proteoforms contain one phosphate moiety. The site-specific annotation of the 5 glycosylation sites present on fetuin (A = Asn156, B = Asn176, C = Thr256, D = Thr270, and E = Ser346) was assigned by using our in-house developed software, making use of the integration of the native MS and peptide-centric MS data. The complete list with all annotated proteoforms and their relative abundance can be found supplemental Table S2.
Fig. 4.
Fig. 4.
Classification of fetuin proteoform profiles by hierarchical clustering. A, Unsupervised clustering of fetuin proteoform profiles derived from the 10 healthy individuals based on the correlation between their native mass spectra. B, Clustering of fetuin proteoform profiles derived from 10 healthy individuals based on specific signature peaks (see supplemental Fig. S4). Color and size of the circles represent the similarity between the proteoform profiles of different genotypes. Orange, blue and green boxes represent fetuin originating from donors representing the AHSG*2, AHSG*1 and AHSG1/2 genotypes, respectively. Comparing the results in A and B the classification in A is distorted by two outliers derived from donor F5 (AHSG*1) and M5 (AHSG*2), respectively caused by a major change in fetuin phosphorylation in these two individuals. C, Clustering of fetuin proteoform profiles derived from 10 healthy and 10 septic patients based on specific signature peaks, where pink dashed line boxes indicate fetuin derived from septic individuals.
Fig. 5.
Fig. 5.
Septic patients display enhanced fucosylation on Asn176. A, Two representative zero-charge deconvoluted native mass spectra of a healthy donor (F1) and septic patient (F6). The average intensities of the site-specifically assigned fucosylated proteoform and its non-fucosylated variant were used for the determination of the relative fucosylation level. The intensities were extracted from the native mass spectra using the corresponding ion signals detected in the 12+ and 13+ charge states. B, Comparison of the extent of fucosylation obtained from the native mass spectra and peptide-centric analysis. The level of fucosylation in the peptide-centric data was determined by ratios of the peak areas of the fucosylated and non-fucosylated peptide containing the sepsis affected N-glycosylation site Asn176. Both approaches resulted in a statistically significant separation of the healthy and septic patients with p values of 0.001 and 0.006, respectively. (A = Asn156, B = Asn176, C = Thr256, D = Thr270, and E = Ser346).
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