Development of a parallel microbore hollow fiber enzyme reactor platform for online 18O-labeling: Application to lectin-specific lung cancer N-glycoproteome

摘要

We introduce a simple online18O-labeling protocol for protein samples that uses a parallelizing microbore hollow fiber enzyme reactor (mHFER) as an alternative tool for online proteolytic digestion. Online18O-labeling is performed by separately attaching two mHFERs in parallel to a 10-port switching valve with a high-pressure syringe pump and two syringes containing16O- or18O-water.16O-/18O-labeled peptides are formed in this manner and simultaneously analyzed online using nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) without any residual trypsin activity. The usefulness of a parallel mHFER platform (P-mHFER) in18O-labeling was tested using both cytochrome C and alpha-1-acid-glycoprotein to verify the incorporation level of two18O atoms into tryptic peptides and to provide a quantitative assessment with varied mixing ratios. Additionally, our18O-labeling approach was used to study the serumN-glycoproteome from lung cancer patients and controls to evaluate the applicability of lectin-based quantitativeN-glycoproteomics. We successfully quantified 76 peptides (from 62N-glycoproteins). Nineteen of these peptides from lung cancer serum were up-/down-regulated at least 2.5-fold compared to controls. As a result, the P-mHFER-based online18O-labeling platform presented here can be a simple and reproducible way to allow quantitative proteomic analysis of diverse proteome samples.