title: MTRAQ-based quantitative analysis combined with peptide fractionation based on cysteinyl peptide enrichment.
abstract: In the present study, the fractionation scheme for cysteinyl peptide enrichment (CPE) was combined with the mass differential tags for relative and absolute quantification (mTRAQ) method to reduce sample complexity and increase proteome coverage. Cysteine residues of the proteins were first alkylated using iodoacetyl PEG2-biotin instead of other conventional alkylating agents such as iodoacetamide. After trypsin digestion, amine groups were labeled with mTRAQ, and these labeled peptides were fractionated according to the presence or absence of cysteine residues using avidin-biotin affinity chromatography. With these approaches, we were able to divide the peptides into the two fractions with more than 90% fractionation efficiency for standard protein and MCF7 cell lysate. When the fractionation strategy was applied to colorectal cancer tissue samples, we were able to obtain quantitative information that was consistent with the previous study based on mTRAQ quantification, implying that the cysteine-based fractionation method does not affect mTRAQ quantification. We expect that the mTRAQ-based quantitative analysis combined with peptide fractionation through the CPE strategy would allow for deep-down analysis of proteome samples and ultimately for increasing proteome coverage with simultaneous quantification for biomarker discovery.
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea; Department of Biological Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea.
authors.forename: Jeonghun
authors.initials: J
authors.lastname: Yeom
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea; Department of Life Sciences, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Min Jung
authors.initials: MJ
authors.lastname: Kang
authors.affiliation: College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea.
authors.forename: Dongyun
authors.initials: D
authors.lastname: Shin
authors.affiliation: Department of Life Sciences, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Hyun Kyu
authors.initials: HK
authors.lastname: Song
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea; Department of Biological Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea. Electronic address: clee270@kist.re.kr.
authors.forename: Cheolju
authors.initials: C
authors.lastname: Lee
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea. Electronic address: jelee9137@kist.re.kr.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
mesh_terms: D003545:Cysteine; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D061986:MCF-7 Cells; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D014357:Trypsin; D003545:Cysteine
keywords: Cysteinyl peptide enrichment; Mass spectrometry; Peptide fractionation; Quantification