This paper introduces a simple, inexpensive, and robust quantitative proteomic method for quantifying N-linked glycoproteins based on isotope-coded carbamidomethylation (iCCM) incorporated into an online microbore hollow fiber enzyme reactor and nanoflow liquid chromatography-tandem mass spectrometry (mHFER-nLC-MS/MS). The iCCM quantitation uses carbamidomethylation (CM; a routine protection of thiol groups before proteolysis) of the Cys residue of proteins with iodoacetamide (IAA) or its isotope (IAA-13C 2,D2: 4 Da difference). CM-/iCCM-labeled proteome samples are mixed for proteolysis; then, online enrichment of N-glycopeptides using lectin affinity is carried out in an mHFER before nLC-MS/MS for quantification using multiple reaction monitoring (MRM). Initial evaluation of the iCCM method varying the mixing ratio of CM-/iCCM-labeled bovine serum albumin (BSA) standards yielded successful quantification of 18 peptides with less than 2% variation in the calculated ratio of light/heavy-labeled peptides. The iCCM quantitation with mHFER-nLC-MS/MS was evaluated with three standard glycoproteins (α-1-acid glycoproteins, fetuin and transferrin) and then applied to serum glycoproteins from liver cancer patients and controls, resulting in successful quantification of 73 N-glycopeptides (from 49 N-glycoproteins), among which 19 N-glycopeptides from 14 N-glycoproteins showed more than a 2.5-fold aberrant change in liver cancer patients' sera compared with the pooled control. Although iCCM quantitation with mHFER-nLC-MS/MS applies only to glycopeptides with Cys residue, the method can offer several advantages over other labeling methods when applied to targeted glycoproteins: The iCCM method does not require an additional labeling reaction under special conditions nor complicated procedures to purify labeled products using additional columns. Isotope labeling at the protein level can minimize potential uncertainty originating from unequal efficiencies in protein digestion in separate vials and retrieval of each labeled peptide when labeling takes place at the peptide level. In addition, the labeling reagents for the iCCM method are readily obtained at a reasonable cost, which can make protein quantification easily accessible.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry