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 18O 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.