Haptoglobin is a liver-secreted glycoprotein with four Nglycosylation sites. Its glycosylation was reported to change in several cancer diseases, which prompted us to examine site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma. To this end, we have used two-dimensional separation composed of hydrophilic interaction and nano-reverse phase chromatography coupled to QTOF mass spectrometry of the enriched glycopeptides. Our results show increased fucosylation of haptoglobin in liver disease with up to six fucoses associated with specific glycoforms of one glycopeptide. Structural analysis using exoglycosidase treatment and MALDI-MS/MS of detached permethylated glycans led to the identification of Lewis Y-type structures observed particularly in the pooled hepatocellular carcinoma sample. To confirm the increase of the Lewis Y structures observed by LC-MS, we have used immunoaffinity detection with Lewis Y-specific antibodies.

N-Glycosylation is a common modification that controls protein folding and multiple functions of mature glycoproteins (1). This co-translational process is controlled by the activity of glycosyltransferases and glycosidases localized in the endoplasmic reticulum and Golgi compartments (2), and its importance increases with complexity of the organism (3, 4). Changes in the abundance and microheterogeneity of glycosylation have been associated with several diseases, including cancer. For example, increased core fucosylation, branching, and terminal sialylation of proteins were associated with carcinogenesis in multiple publications (5-7). In this study, we focus on the quantitative monitoring of changes in N-glycosylation of haptoglobin (Hp) 1 in patients with liver disease. Quantitative changes in protein glycosylation have been analyzed on the level of enzymatically detached N-glycans and on the level of glycopeptides, but the analyses of detached glycans are better developed. Almost all reported quantifications are based on relative quantities partly because quantitative standards are not readily accessible and because changes in relative distributions are considered more important than absolute quantities of the analytes. The only paper we are aware of reporting HPLC-based absolute quantification of glycans used a standard isolated from hen egg yolk for analysis of enzymatically detached N-glycans in rheumatoid arthritis (8).Multiple workflows for relative (semi)quantitative analysis of detached N-glycans, using various analytical techniques, were described (9 -13). Perhaps the most widely used profiling methods are normal phase HPLC of fluorescently labeled glycans (14, 15) and MALDI/TOF mass spectrometric screening of permethylated glycans (16). Chromatographic resolution of the fluorescently labeled complex glycan mixtures can be somewhat limited, although MALDI/TOF screening has From the

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.

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.

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