Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

Abstract

Alternations in the glycosylation of proteins have been described in connection with several cancers, including hepatocellular carcinoma (HCC) and colorectal cancer. Analytical tools, which use combination of liquid chromatography and mass spectrometry, allow precise and sensitive description of these changes. In this study, we use MRM and FT-ICR operating in full-MS scan, to determine ratios of intensities of specific glycopeptides in HCC, colorectal cancer, and liver metastasis of colorectal cancer. Haptoglobin, hemopexin and complement factor H were detected after albumin depletion and the N-linked glycopeptides with fucosylated glycans were compared with their non-fucosylated forms. In addition, sialylated forms of an O-linked glycopeptide of hemopexin were quantified in the same samples. We observe significant increase in fucosylation of all three proteins and increase in bisialylated O-glycopeptide of hemopexin in HCC of hepatitis C viral (HCV) etiology by both LC-MS methods. The results of the MRM and full-MS scan FT-ICR analyses provide comparable quantitative readouts in spite of chromatographic, mass spectrometric and data analysis differences. Our results suggest that both workflows allow adequate relative quantification of glycopeptides and suggest that HCC of HCV etiology differs in glycosylation from colorectal cancer and liver metastasis of colorectal cancer. Significance The article compares N- and O-glycosylation of several serum proteins in different diseases by a fast and easy sample preparation procedure in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry. The results show successful glycopeptides relative quantification in a complex peptide mixture by the high resolution instrument and the detection of glycan differences between the different types of cancer diseases. The presented method is comparable to conventional targeted MRM approach but allows additional curation of the data.

Serum glycoprotein markers in non-alcoholic steato-hepatitis and hepatocellular carcinoma

“…Benicky and coworkers found that the ratios of fucosylated to non-fucosylated forms of the same glycan at amino acid residues 217, 882, 911 and 1029 61 were higher in HCC as compared to controls. Darebna and coworkers observed higher core fucosylation levels at amino acid position 882 55 in HCC as compared to controls, and our findings confirm these results. In addition, we found that the normalized abundance of core fucosylation is statistically significantly higher in NASH and in HCC, as compared to healthy controls.…”

Comparison of Different HILIC Stationary Phases in the Separation of Hemopexin and Immunoglobulin G Glycopeptides and Their Isomers

“…Alterations in glycosylation patterns affect many biological processes, such as protein folding, intracellular sorting, secretion, and host-microbial recognition [ 4 , 5 ]. In addition, abnormal protein glycosylation has been used as a diagnostic tool for many cancers, including breast, prostate, and ovarian cancer [ 6 , 7 , 8 , 9 ]. Yet, despite recent bioanalytical advances in glycomics, glycoprotein analysis remains a challenge.…”

“…Changes in glycopeptide isomeric structures can also be used as potential biomarkers [ 9 , 22 ]. As such, Huang et al have used HILIC to separate sialylated N -glycan isomers of fetuin differing only in α2–3 and α2–6 linkages [ 19 ].…”

Calculating Glycoprotein Similarities From Mass Spectrometric Data

“…The following discussion is oriented toward the use of MS reaction monitoring for protein similarity measurement. A number of groups have published MRM/PRM methods for quantification of glycopeptides ( 47 , 48 , 49 , 50 ). Because peptide backbone dissociation results in low-abundance product ions, the tandem MS transitions often employ neutral saccharide losses from the precursor and oxonium ions.…”