title: Affinity selection of histidine-containing peptides using metal chelate methacrylate monolithic disk for targeted LC-MS/MS approach in high-throughput proteomics.
abstract: 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 efficient 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.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, India.
authors.forename: Rajasekar R
authors.initials: RR
authors.lastname: Prasanna
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, India.
authors.forename: Sinash
authors.initials: S
authors.lastname: Sidhik
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, India.
authors.forename: Agamudi S
authors.initials: AS
authors.lastname: Kamalanathan
authors.affiliation: Agilent Technologies India Pvt. Ltd., Hyderabad, India.
authors.forename: Krishna
authors.initials: K
authors.lastname: Bhagavatula
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, India. Electronic address: indviji70@gmail.com.
authors.forename: Mookambeswaran A
authors.initials: MA
authors.lastname: Vijayalakshmi
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D006639:Histidine / Q000032:analysis* / Q000737:chemistry; D008689:Methacrylates / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008689:Methacrylates; D010446:Peptide Fragments; D006639:Histidine; D003300:Copper
keywords: Bottom-up approach; CIM monolithic disk; Enrichment; Histidine-peptides; Immobilized metal affinity chromatography (IMAC); Targeted affinity selection