title: MS/MS of synthetic peptide is not sufficient to confirm new types of protein modifications.
abstract: Protein post-translational modification (PTM) is one of the major regulatory mechanisms that fine-tune protein functions. Undescribed mass shifts, which may suggest novel types of PTMs, continue to be discovered because of the availabilities of more sensitive mass spectrometry technologies and more powerful sequence alignment algorithms. In this study, the histone extracted from HeLa cells was analyzed using an approach that takes advantages of in vitro propionylation, efficient peptide separation using isoelectric focusing fractionation, and the high sensitivity of the linear ion trap coupled with hybrid FT mass spectrometer. One modified peptide was identified with a new type of protein modification (+42 Da), which was assigned to acetylation of threonine 15 in histone2A. The modified peptide was verified by careful manual evaluation of the tandem mass spectrum and confirmed by high-resolution MS/MS analysis of the corresponding synthetic peptide. However, HPLC coelution and MS/MS/MS of key ions showed that the +42 Da mass shifts at threonine residue did not correspond to acetylation. The key fragment ion, y4, in the MS/MS/MS spectra (indicative of the modification site) differed between the in vivo and synthetic peptide. We showed that the misidentification was originated from sequence homologues and chemical derivitization during sample preparation. This result indicated that a more stringent procedure that includes MS/MS, MS/MS/MS, and HPLC coelution of synthetic peptides is required to identify a new PTM.
authors.affiliation: College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Korea. sangkyu@knu.ac.kr
authors.forename: Sangkyu
authors.initials: S
authors.lastname: Lee
authors.forename: Minjia
authors.initials: M
authors.lastname: Tan
authors.forename: Lunzhi
authors.initials: L
authors.lastname: Dai
authors.forename: Oh Kwang
authors.initials: OK
authors.lastname: Kwon
authors.forename: Jeong Soo
authors.initials: JS
authors.lastname: Yang
authors.forename: Yingming
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006657:Histones / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis; D011422:Propionates / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006657:Histones; D010455:Peptides; D011422:Propionates; D013912:Threonine; D012694:Serine; C029658:propionic acid