PRM-MS Quantitative Analysis of Isomeric N-Glycopeptides Derived from Human Serum Haptoglobin of Patients with Cirrhosis and Hepatocellular Carcinoma

Abstract

Currently, surveillance strategies have inadequate performance for cirrhosis and early detection of hepatocellular carcinoma (HCC). The glycosylation of serum haptoglobin has shown to have significant differences between cirrhosis and HCC, thus can be used for diagnosis. We performed a comprehensive liquid chromatography—parallel reaction monitoring—mass spectrometry (LC-PRM-MS) approach, where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system, to study the site-specific isomerism of haptoglobin (Hp) extracted from cirrhosis and HCC patients. We found that our strategy was able to identify a large number of isomeric N-glycopeptides, mainly located in the Hp glycosylation site Asn207. Four N-glycopeptides were found to have significant changes in abundance between cirrhosis and HCC samples (p < 0.05). Strategic combinations of the significant N-glycopeptides, either with alpha-fetoprotein (AFP) or themselves, better estimate the areas under the curve (AUC) of their respective receiver operating characteristic (ROC) curves with respect to AFP. The combination of AFP with the isomeric sialylated fucosylated N-glycopeptides Asn207 + 5-6-1-2 and Asn207 + 5-6-1-3, resulted with an AUC value of 0.98, while the AUC value for AFP alone was 0.85. When comparing cirrhosis vs. early HCC, the isomeric N-glycopeptide Asn207 + 5-6-0-1 better estimated AUC with respect to AFP (AUCAFP = 0.81, and AUCAsn207 + 5-6-0-1 = 0.88, respectively).

Glycopeptides with Sialyl Lewis Antigen in Serum Haptoglobin as Candidate Biomarkers for Nonalcoholic Steatohepatitis Hepatocellular Carcinoma Using a Higher-Energy Collision-Induced Dissociation Parallel Reaction Monitoring-Mass Spectrometry Method

“…It was further shown that when AFP was combined with glycopeptides N207_A3G3F1S2 and N207_A3G3F1S2 as a 3-marker panel, the result provided a better AUC value than AFP alone. 46 Different from this work, the current work contains a total of 95 patients’ serum samples in a larger sample set and the precursor ions selected in our current experiment were based on our previous work 22 , 23 where both charge states 3 and 4 precursor ions were included. These differences would provide more accurate and precise results, which include significant changes of glycopeptides in abundance involving sites N184 and N241.…”

“…In a recent work, it has been reported that four glycopeptides involving site N207 of Hp changes significantly in abundance between HCC and cirrhosis from a total of 30 patients’ serum samples. It was further shown that when AFP was combined with glycopeptides N207_A3G3F1S2 and N207_A3G3F1S2 as a 3-marker panel, the result provided a better AUC value than AFP alone . Different from this work, the current work contains a total of 95 patients’ serum samples in a larger sample set and the precursor ions selected in our current experiment were based on our previous work , where both charge states 3 and 4 precursor ions were included.…”

Glycomic and Glycoproteomic Techniques in Neurodegenerative Disorders and Neurotrauma: Towards Personalized Markers

“…With the aforementioned dissociation techniques, several acquisition strategies can be used to improve the quantitation of glycans and glycopeptides. Multiple reaction monitoring (MRM) and recently parallel reaction monitoring (PRM) have been demonstrated to be accurate targeted quantitation methods for both glycans and glycopeptides [178][179][180][181]. The limitation of MRM and PRM is that they cannot be used for untargeted analysis.…”

MS-based glycomics: An analytical tool to assess nervous system diseases

“…Another advantage of PRM is that it enables the monitoring of all fragment ions simultaneously, thus allowing the selection of any fragments for quantitation. The MRM for glycomic analysis has been demonstrated ( Zhou et al, 2015 ), while PRM has been proved in a glycoproteomic study ( Gutierrez Reyes et al, 2021 ). The pitfall of both MRM and PRM is that they cannot be utilized in untargeted glycomic analysis, thus limiting their use in initial glycomic studies of CNS diseases.…”