Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS

Abstract

Intact N-glycopeptide analysis remains challenging due to the complexity of glycopeptide structures, low abundance of glycopeptides in protein digests, and difficulties in data interpretation/quantitation. Herein, we developed a workflow that involved advanced methodologies, the EThcD- MS/MS fragmentation method and data interpretation software, for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis. Haptoglobin was immunopurified from 20 μL of serum in patients with early HCC, liver cirrhosis, and healthy controls, respectively, followed by trypsin/GluC digestion, glycopeptide enrichment, and LC-EThcD-MS/MS analysis. Identification and differential quantitation of site-specific N-glycopeptides were performed using a combination of Byonic and Byologic software. In total, 93, 87, and 68 site-specific N-glycopeptides were identified in early HCC, liver cirrhosis, and healthy controls, respectively, with high confidence. The increased variety of N-glycopeptides in liver diseases compared to healthy controls was due to increased branching with hyper-fucosylation and sialylation. Differential quantitation analysis showed that 5 site-specific N-glycopeptides on sites N184 and N241 were significantly elevated in early HCC compared to cirrhosis (p < 0.05) and normal controls (p ≤ 0.001). The result demonstrates that the workflow provides a strategy for detailed profiles of N-glycopeptides of patient samples as well as for relative quantitation to determine the level changes in site-specific N-glycopeptides between disease states.

Glycoproteomic markers of hepatocellular carcinoma‐mass spectrometry based approaches

“…This software is also an important advance because manual analysis would be difficult and time‐consuming. The analysis of patient serum resulted in 279 N‐glycopeptide spectral matches that corresponded to 98 site‐specific N‐glycopeptides (Zhu et al, ). In addition, several key structures were found to be quantitatively different between early stage HCC and cirrhosis, including a bifucosylated tetra‐antennary form reported in earlier work (Zhu et al, ).…”

“…A recent strategy to analyze intact glycopeptides with mass spectrometry developed a quantitative EThcD‐MS/MS method to determine changes in intact N ‐glycopeptides between early HCC and liver cirrhosis for Hp in patient serum (Zhu et al, ). In this work, Hp was immunopurified from 20 µL of serum followed by digestion with trypsin and GluC, glycopeptide enrichment with HILIC TopTips, and LC‐EThcD‐MS/MS analysis on an Orbitrap Fusion Lumos Tribrid mass spectrometer.…”

N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases

“…TPLTAN 205 ITK (H5N5S1F1) may represent this N-glycosylation site attached with the monosialylated bisected fucosylated biantennary oligosaccharide and TPLTAN 205 ITK (H5N4S2F1) represents this site attached with the fucosylated biantennary fully sialylated oligosaccharide. Interestingly, increases in fucosylation and sialylation have been observed in patients with HCC (29,30). Likewise, increased glycosylation such as carbohydrate antigen 19 -9 was commonly elevated in the serum of patients with a variety of cancers, including pancreatic, gastric, and colorectal cancers (31).…”

Optimal Dissociation Methods Differ for N- and O-glycopeptides

“…15 To address this issue, hybrid methods that use vibrational activation to provide supplemental energy for ETD reactions have emerged and gained traction in glycoproteomics, with the most popular being ETD followed by supplemental HCD (EThcD). [33][34][35][36][37][38][39][40][41][42][43][44][45] Even so, HCD remains widely used in N-glycoproteomics. [46][47][48][49][50][51][52][53][54][55][56][57] Tryptic Nglycopeptides tend to harbor only one potential glycosite, as defined by its sequon N-X-S/T, where X represents any amino acid other than proline, which limits dependence on peptide fragments that retain intact glycans.…”