Abstract
A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.
Highlights
From the ‡Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, China, §The International Cooperation Laboratory on Signal Transduction of Eastern Hepatobiliary Surgery Institute, Second Military Medical University, Shanghai, 200438, China, ¶Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, 200433, China
The nonglycopeptides of captured glycoproteins were released with trypsin digestion and the digest was analyzed with two-dimensional liquid chromatography (LC)-Mass spectrometry (MS)/MS
Great technical achievements have been made in the past few years to measure the global protein expression with mass spectrometry based quantitative proteomics approach, there are still some unresolved issues for the highthroughput and accurate quantification of proteins with modifications like glycosylation
Summary
A depletion of some abundant proteins before lectin capture may reduce nonspecific adsorption and enhance analytical performance [12], this procedure may deplete some proteins of low abundance, which are possible potential biomarkers because it has been found that immunosorbent columns and antibodies would bind to more than 100 proteins nonspecifically, some of which are glycosylated [13]. Another popular method for the isolation, identification, and quantification of glycoprotein is the capture of oxidized glycopeptides by hydrazide chemistry [14]. Because there exits great heterogeneity in the occupancy of different N-glycosites in glycoproteins with multiple glycosylated sites, quantification of glycoprotein by glycopeptides would lead to ambiguous results
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