title: Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry.
abstract: Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang 363-883, Republic of Korea; Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Kwang Hoe
authors.initials: KH
authors.lastname: Kim
authors.affiliation: Department of Biomedical Science, Cheongju University, Cheongju 363-764, Republic of Korea.
authors.forename: Yeong Hee
authors.initials: YH
authors.lastname: Ahn
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang 363-883, Republic of Korea.
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Ji
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang 363-883, Republic of Korea.
authors.forename: Ju Yeon
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang 363-883, Republic of Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: An
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang 363-883, Republic of Korea; Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea. Electronic address: jongshin@kbsi.re.kr.
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D002138:Calibration; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000345:Affinity Labels; D001798:Blood Proteins; D010455:Peptides
keywords: Hybrid quadrupole time-of-flight mass spectrometer; Low-abundance serological protein; Peptide affinity-based enrichment; Pseudo-multiple reaction monitoring