4 research outputs found
Data Independent Analysis of IgG Glycoforms in Samples of Unfractionated Human Plasma
Glycosylation
regulates functional responses mediated by the interaction
of IgG with their receptors. Multiple analytical methods have been
designed for the determination of the IgG N-glycan microheterogeneity,
including MS methods for the analysis of site specific glycoforms
of IgG. However, measurement of low abundant glycoforms remains challenging
in complex samples like serum without enrichment of the IgG. We present
a workflow for quantitative analysis of site specific glycoforms of
IgG based on data independent acquisition (DIA) of Y-ions generated
under “minimal” fragmentation conditions. The adjusted
collision induced dissociation (CID) conditions generate specific
Y-ions in the yield of up to 60% precursor ion intensity. These selective
fragments, measured in high resolution, improve specificity of detection
compared to the typically quantified B-ions which have higher overall
intensity but lower signal-to-noise ratios. Under optimized conditions,
we achieve label-free quantification of the majority of previously
reported glycoforms of IgG (26 glycoforms of IgG1, 22 glycoforms of
IgG 2/3, and 19 glycoforms of IgG4) directly in unfractionated samples
of human plasma and we detect traces of previously unreported glycoforms
of IgG1, including doubly fucosylated glycoforms. The SWATH data independent
quantification of IgG glycoforms in pooled plasma samples of patients
with liver cirrhosis detects reliably the expected changes in the
quantity of major glycoforms compared to healthy controls. Our results
show that optimized CID fragmentation enables DIA of IgG glycoforms
and suggest that such workflow may enable quantitative analyses of
the glycoproteome in complex matrixes
Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease
Enhanced fucosylation has been suggested
as a marker for serologic
monitoring of liver disease and hepatocellular carcinoma (HCC). We
present a workflow for quantitative site-specific analysis of fucosylation
and apply it to a comparison of hemopexin (HPX) and complement factor
H (CFH), two liver-secreted glycoproteins, in healthy individuals
and patients with liver cirrhosis and HCC. Label-free LC-MS quantification
of glycopeptides derived from these purified glycoproteins was performed
on pooled samples (2 pools/group, 5 samples/pool) and complemented
by glycosidase assisted analysis using sialidase and endoglycosidase
F2/F3, respectively, to improve resolution of glycoforms. Our analysis,
presented as relative abundance of individual fucosylated glycoforms
normalized to the level of their nonfucosylated counterparts, revealed
a consistent increase in fucosylation in liver disease with significant
site- and protein-specific differences. We have observed the highest
microheterogeneity of glycoforms at the N187 site of HPX, absence
of core fucosylation at N882 and N911 sites of CFH, or a higher degree
of core fucosylation in CFH compared to HPX, but we did not identify
changes differentiating HCC from matched cirrhosis samples. Glycosidase
assisted LC-MS-MRM analysis of individual patient samples prepared
by a simplified protocol confirmed the quantitative differences. Transitions
specific to outer arm fucose document a disease-associated increase
in outer arm fucose on both bi- and triantennary glycans at the N187
site of HPX. Further verification is needed to confirm that enhanced
fucosylation of HPX and CFH may serve as an indicator of premalignant
liver disease. The analytical strategy can be readily adapted to analysis
of other proteins in the appropriate disease context
Protein and Site Specificity of Fucosylation in Liver-Secreted Glycoproteins
Chronic liver diseases are a serious
health problem worldwide.
One of the frequently reported glycan alterations in liver disease
is aberrant fucosylation, which was suggested as a marker for noninvasive
serologic monitoring. We present a case study that compares site specific
glycoforms of four proteins including haptoglobin, complement factor
H, kininogen-1, and hemopexin isolated from the same patient. Our
exoglycosidase-assisted LC–MS/MS analysis confirms the high
degree of fucosylation of some of the proteins but shows that microheterogeneity
is protein- and site-specific. MSn analysis of permethylated detached
glycans confirms the presence of LeY glycoforms on haptoglobin, which
cannot be detected in hemopexin or complement factor H; all three
proteins carry Lewis and H epitopes. Core fucosylation is detectable
in only trace amounts in haptoglobin but with confidence on hemopexin
and complement factor H, where core fucosylation of the bi-antennary
glycans on select glycopeptides reaches 15–20% intensity. These
protein-specific differences in fucosylation, observed in proteins
isolated from the same patient source, suggest that factors other
than up-regulation of enzymatic activity regulate the microheterogeneity
of glycoforms. This has implications for selection of candidate proteins
for disease monitoring and suggests that site-specific glycoforms
have structural determinants, which could lead to functional consequences
for specific subsets of proteins or their domains
Site-Specific Glycan Microheterogeneity of Inter-Alpha-Trypsin Inhibitor Heavy Chain H4
Inter-alpha-trypsin
inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein
produced primarily in the liver, secreted into the blood, and identified
in serum. ITIH4 is involved in liver development and stabilization
of the extracellular matrix (ECM), and its expression is altered in
liver disease. In this study, we aimed to characterize glycosylation
of recombinant and serum-derived ITIH4 using analytical mass spectrometry.
Recombinant ITIH4 was analyzed to optimize glycopeptide analyses,
followed by serum-derived ITIH4. First, we confirmed that the four
ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated
by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence
of <sup>18</sup>O water. Next, we performed glycosidase-assisted LC–MS/MS
analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic
interaction liquid chromatography to characterize ITIH4 N-glycoforms.
While microheterogeneity of N-glycoforms differed between ITIH4 protein
expressed in HEK293 cells and protein isolated from serum, occupancy
of N-glycosylation sites did not differ. A fifth N-glycosylation site
was discovered at N274 with the rare nonconsensus NVV motif. Site
N274 contained high-mannose N-linked glycans in both serum and recombinant
ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and
documented that utilization of O-glycosylation sites on ITIH4 differed
between the cell line and serum