Affinity selection of histidine-containing peptides using metal chelate methacrylate monolithic disk for targeted LC-MS/MS approach in high-throughput proteomics

摘要

In recent years, bottom-up approach has become the popular method of choice for large scale analysis of complex proteome samples. Peptide fractionation determines the efficiency of the bottom-up method and often the resolving power of reverse phase liquid chromatography (RPLC) is insufficient for effi-cient protein identification in case of complex biological samples. To overcome the inherent limitation of proteomics associated with sample complexity, we evaluated fast flow metal chelate methacrylate monolithic system - CIM (Convective Interaction Media) disk chelated with Cu(II) for targeted affinity selection of histidine-containing peptides. Initially the Cu(II)-IMAC using CIM disk was evaluated using tryptic digest of protein mixtures of 8 model proteins and was found to be highly efficient in capturing His-containing peptides with high degree of specificity and selectivity. Further the efficiency of His-peptide enrichment using CIM-IMAC was also demonstrated using complex biological samples like total Escherichia coli cell lysate. The analysis of the Cu(II)-IMAC retained peptides from tryptic digests of model protein mixture and E. coli not only demonstrated a significant reduction in sample complexity but also subsequently enabled the identification of additional peptides. His-peptide enrichment also enabled the identification of low abundant proteins that were not detected in the analysis of total E. coli digest.