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.

Nonalcoholic steatohepatitis (NASH) is rising in prevalence in the United States and is a growing cause of hepatocellular carcinomas (HCCs). Site-specific glycan heterogeneity on glycoproteins has been shown as a potential diagnostic biomarker for HCC. Herein, we have performed a comprehensive screening of site-specific N-glycopeptides in serum haptoglobin (Hp), a reporter molecule for aberrant glycosylation in HCC, to characterize glycopeptide markers for NASH-related HCCs. In total, 70 NASH patients (22 early HCC, 15 advanced HCC, and 33 cirrhosis cases) were analyzed, with Hp purified from 20 μL of serum in each patient, and 140 sets of mass spectrometry (MS) data were collected using liquid chromatography coupled with electron-transfer high-energy collisional dissociation tandem MS (LC-EThcD-MS/MS) for quantitative analysis on a novel software platform, Byos. Differential quantitation analysis revealed that five N-glycopeptides at sites N184 and N241 were significantly elevated during the progression from NASH cirrhosis to HCC (p < 0.05). Receiver operating characteristic (ROC) curve analysis demonstrated that the N-glycopeptides at sites N184 and N241 bearing a monofucosylated triantennary glycan A3G3F1S3 had the best diagnostic performance in detection of early NASH HCC, area under the curve (AUC) = 0.733 and 0.775, respectively, whereas α-fetoprotein (AFP) had an AUC of 0.692. When combined with AFP, the two panels improved the sensitivity for early NASH HCC from 59% (AFP alone) to 73% while maintaining a specificity of 70%, based on the optimal cutoff. Two-dimensional (2-D) scatter plots of the AFP value and N-glycopeptides showed that these N-glycopeptide markers detected 58% of AFP-negative HCC patients as distinct from cirrhosis. These site-specific N-glycopeptides could serve as potential markers for early detection of HCC in patients with NASH-related cirrhosis.

Changes in N-glycosylation on specific peptide sites of serum proteins have been investigated as potential markers for diagnosis of nonalcoholic steatohepatitis (NASH)-related HCC. To accomplish this work, a novel workflow involving broad-scale marker discovery in serum followed by targeted marker evaluation of these glycopeptides were combined. The workflow involved an LC-Stepped HCD-DDA-MS/ MS method coupled with offline peptide fractionation for large-scale identification of N-glycopeptides directly from pooled serum samples (each n = 10) as well as differential determination of N-glycosylation changes between disease states. We then evaluated several potentially diagnostic N-glycopeptides among 78 individual patient samples (40 cirrhosis, 28 early stage NASH HCC, and 10 late-stage NASH HCC) by LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 65 targeted glycopeptides from 7 glycoproteins. Of these targets, we found site-specific N-glycopeptides n 169 GSLFAFR_HexNAc(4)Hex(5)-NeuAc(2) and n 242 ISDGFDGIPDNVDAALALPAHSYSGR_HexNAc(5)Hex( 6)Fuc(1)NeuAc(3) from VTNC were significantly increased comparing samples from patients with NASH cirrhosis and NASH HCC (p < 0.05). When combining results of these 2 glycopeptides with AFP, the ROC curve analysis demonstrated the AUC value increased to 0.834 (95% CI, 0.748−0.921) and 0.847 (95% CI, 0.766−0.932), respectively, as compared to that of AFP alone (AUC = 0.791, 95% CI, 0.690−0.892). These 2 glycopeptides may serve as potential biomarkers for early HCC diagnosis in patients with NASH related cirrhosis.

Serum-derived extracellular vesicles (EVs) are a promising source of biomarkers; however, major challenges in EV separation and proteomic profiling remain for isolating EVs from a small amount, that is, on the microliter scale, of human serum while minimizing the contamination of blood proteins and lipoprotein particles coeluting in EV preparations. Herein we have developed a column-based CD9-antibody-immobilized high-performance liquid chromatography immunoaffinity chromatography(CD9-HPLC-IAC) technology for EV isolation from a microliter scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 μL of serum in 30 min with a yield of 8.0 × 10 9 EVs, where EVs were further processed with a postcolumn cleaning step using the 50 kDa molecular weight cut-off filter for the buffer exchange, concentration, and reduction of potentially coeluting serum proteins. In total, 482 proteins were identified in EVs by using liquid chromatography tandem mass spectrometry, including the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101. The statistical analysis of EV protein content showed that the top 10 serum proteins in EVs were significantly decreased by using the CD9-HPLC-IAC method compared with the use of ultracentrifugation (p = 0.001) and size exclusion chromatography (p = 0.009), and apolipoproteins were significantly reduced 4.8-fold compared with the SEC method (p < 0.001). The result demonstrates the potential of the CD9-HPLC-IAC method for the efficient isolation and proteomic characterization of EVs from a microscale volume of serum.

Nonalcoholic steatohepatitis (NASH) is the fastest growing cause of hepatocellular carcinoma (HCC) in the United States. Changes in N -glycosylation on specific glycosites of serum proteins have been investigated as potential markers for the early detection of NASH-related HCC. Herein, we report a glycopeptide with a Sialyl Lewis structure derived from serum haptoglobin (Hp) as a potential marker for NASH related HCCs among 95 patients with NASH, including 46 cirrhosis, 32 early-stage HCC, and 17 late-stage HCC. Hp immuno-isolated from patient serum was analyzed using LC-HCD-PRM-MS/MS followed by data analysis via Skyline software. Two glycopeptides involving site N184 and four glycopeptides involving site N241 were significantly changed in patients with HCC vs NASH cirrhosis ( P < 0.05). The two-marker panel using N -glycopeptide N241_A4G4F2S4 showed the best performance for HCC detection when combined with α-fetoprotein (AFP), with an improved estimated area under the curve (AUC) = 0.898 (95% CI: 0.835, 0.951), compared to the AUC of 0.790(95% CI, 0.697 0.872) using AFP alone ( P = 0.048). At 90% specificity, the combination of N241_A4G4F2S4 + AFP had an improved sensitivity of 63.3%, compared to the sensitivity of 52.3% using AFP alone. When using three markers, the panel of AFP + N241_A2G2F1S2 + N241_A4G4F2S4 yielded an estimated AUC of 0.928 (95% CI: 0.877, 0.970). Our findings indicated that N241_A4G4F2S4 may play an important role in distinguishing HCC from NASH cirrhosis.