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).
Highlights
To characterize the alterations in site-specific glycopeptide forms toglobin was purified from a 20 μL aliquot of serum from each patient, by using an HPLCbetween cirrhosis and hepatocellular carcinoma (HCC)
Since our LC-parallel reaction monitoring (PRM)-MS approach provided clear differences in site-specific N-glycopepIn addition, the important number of isomeric N-glycopeptides resolved by our LC-PRMtides between cirrhosis and HCC, we further focused on investigating the differentially
We revealed the site-specific microheterogeneity of serum haptoglobin, and demonstrated that the developed isomeric N-glycopeptides can be used to differentiate nonalcoholic steatohepatitis (NASH) cirrhosis and NASH-related HCC
Summary
The development of accurate markers for the early stages of cancer types with high mortality rates, such as hepatocellular carcinoma (HCC) [4], may provide opportunities for more effective patient treatments [5]. HCCs develop in the setting of cirrhosis [6]; current early detection strategies, using abdominal ultrasound and alpha-fetoprotein (AFP), have inadequate sensitivity and specificity for early detection [7]. The performance of ultrasound is further diminished in patients with nonalcoholic steatohepatitis (NASH), associated with central obesity, and the fastest growing cause of HCC in the US [8]. An accurate marker-based strategy, in patients with NASH, has the potential to improve HCC-related outcomes
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