Development of new stable isotope labeling method for quantitative proteomics

다국어 초록 (Multilingual Abstract)

In the study, we introduce an isotope-coded caramidomehtylation (iCCM) so as to be novel, facile, inexpensive, and reproducible, isotope labeling strategy for quantitative proteomics. In iCCM, isotope-labeling for protein samples is performed by means of incorporating each of iodoacetamide (IAA) for CM and its isotope (IAA-13C2, D2) for iCCM into all cysteine residues upon protein(s) during general alkylation procedure, thereby leading to mass shift of all cysteine residues to be + 4 Da. We evaluated the efficiency of iCCM method with BSA by varying mixing ratios. Furthermore, we also compared mTRAQ, as a conventional non-isobaric labeling method, with that of iCCM developed in this study. To examine the efficiency in quantitative proteomics, we used five protein standards (i.e., bovine serum albumin, serotransferrin, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin), and followed by which the resulting peptides labeled separately with each of CM and iCCM were analyzed using a liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS). From our experiments, we found that iCCM-based labeling is superior to that of mTRAQ in both of the sequence coverage of 5 protein standards and reproducibility in the observed ratios of the corresponding peptides. To expand the potential of iCCM onto discovering cancer-specific N-glycoprotein markers, we used a microbore hollow fiber enzyme reactor (mHFER)-nLC-ESI-MS/MS that enables either of online proteoyltic digestion or lectin-specific N-glycoprotein enrichment of those N-glycopeptides (i.e., 65 ConA-specific, 40 SNA-specific, and 49 AAL-specific glycopeptides), we found that 11 N-glycopeptides were specifically up-/down-regulated from lung cancer sera, compared to that of controls.