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 the early detection of hepatocellular carcinoma (HCC). Protein glycosylation is a potential source of biomarkers to differentiate between cirrhosis and HCC. We performed a comprehensive LC-PRM-MS approach where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system to study the microheterogeneity of haptoglobin (Hp) extracted from 15 patients with cirrhosis and 15 with HCC. We found that our strategy was able to identify a large number of isomeric N-glycopeptides mainly located in the glycosylation site Asn207. Nine out of twelve N-glycopeptides, located in the Asn207 site, had significant differences in abundance between patients with cirrhosis and HCC (p < 0.05). The area under the curve (AUC) of alpha-fetoprotein (AFP) was alone 0.85, which improved to 0.95 (95% CI: 0.88, 1) when NLF_5613 Isomer 1 was combined with AFP. When comparing the early HCC vs. cirrhosis, four sialylated-fucosylated glycopeptides better estimated AUCs with respect to AFP (AUCAFP = 0.66, and AUCN−glycopeptides = 0.86, 0.84, 0.88, and 0.80, respectively). Further large scale validation of glycopeptides for the early detection of HCC is warranted.

MS‐based glycomics and glycoproteomics methods enabling isomeric characterization

“…Whereas advanced fragmentation techniques such as high energy collision dissociation (HCD), electron transfer/higher‐energy collision dissociation (EThcD), electron‐detachment dissociation (EDD), infrared multiphoton dissociation (IRMPD), and UV‐photon dissociation (UVPD) can provide sufficient fragment ions, and therefore structural information of large molecules as are glycopeptides (Depraz Depland et al, 2018; Myers et al, 2013, Riley & Coon, 2018; Zhurov et al, 2013). Besides, targeted‐MS n modes such as MRM and PRM provide an increase in specificity and sensitivity that can be extrapolated to accurate quantitation methods of isomeric glycan and glycopeptides (Zhou et al, 2015; Reyes et al, 2021).…”

“…Gutierrez Reyes et al recently reported a comprehensive LC‐PRM‐MS approach where a targeted parallel reaction monitoring (PRM) strategy was used for the quantitation of isomeric haptoglobin N ‐glycopeptides derived from cirrhosis and hepatocellular carcinoma (HCC) samples (Reyes et al, 2021). The target glycopeptides were acquired and fragmented by HCD with a collision energy of 25%.…”

“…PGC columns have proved to be precious tools to achieve isomeric separation of both native as well as permethylated glycans (Ashwood et al, 2019; Gautam et al, 2020; Peng et al, 2019; Veillon et al, 2017; Zhou et al, 2016). Unlike C18, PGC columns demonstrate stronger retention for glycans and can also retain small glycans like O‐ glycans.…”

“…Increasing temperature in RPLC enables the enhancement of resolution for both sialylated O‐ and N‐ glycopeptides. Adopting this study, Gutierrez Reyes et al (2021) combined a targeted parallel reaction monitoring (PRM) with C18‐LC for a comprehensive study on the microheterogeneity of haptoglobin. A total of 73 isomeric structures from 42 N‐ glycopeptides from sites 184 Asn, 207 Asn, and 241 Asn were identified and quantified.…”

“…LC-MS has proven to be extremely useful for the separation of complex isomeric glycan and glycopeptide structures. Unfortunately, the unavailability of standards complicates the characterization of the resolved isomers by using orthogonal derivation or enzymatic strategies (Gutierrez Reyes et al, 2021;Nishikaze et al, 2017;. The applications of MS n methods have demonstrated to assign structural features independent of standards due to the predictability of fragmentation patterns.…”