title: Improved proteomic analysis pipeline for LC-ETD-MS/MS using charge enhancing methods.
abstract: Electron transfer dissociation (ETD) is a useful and complementary activation method for peptide fragmentation in mass spectrometry. However, ETD spectra typically receive a relatively low score in the identifications of 2+ ions. To overcome this challenge, we, for the first time, systematically interrogated the benefits of combining ion charge enhancing methods (dimethylation, guanidination, m-nitrobenzyl alcohol (m-NBA) or Lys-C digestion) and differential search algorithms (Mascot, Sequest, OMSSA, pFind and X!Tandem). A simple sample (BSA) and a complex sample (AMJ2 cell lysate) were selected in benchmark tests. Clearly distinct outcomes were observed through different experimental protocol. In the analysis of AMJ2 cell lines, X!Tandem and pFind revealed 92.65% of identified spectra; m-NBA adduction led to a 5-10% increase in average charge state and the most significant increase in the number of successful identifications, and Lys-C treatment generated peptides carrying mostly triple charges. Based on the complementary identification results, we suggest that a combination of m-NBA and Lys-C strategies accompanied by X!Tandem and pFind can greatly improve ETD identification.
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, People's Republic of China. luhaojie@fudan.edu.cn
authors: Li-Qi
authors: LQ
authors: Xie
authors: Cheng-Pin
authors: CP
authors: Shen
authors: Min-Bo
authors: MB
authors: Liu
authors: Zhong-Da
authors: ZD
authors: Chen
authors: Ru-Yun
authors: RY
authors: Du
authors: Guo-Quan
authors: GQ
authors: Yan
authors: Hao-Jie
authors: HJ
authors: Lu
authors: Peng-Yuan
authors: PY
authors: Yang
mesh_terms: D000465:Algorithms; D000818:Animals; D001592:Benzyl Alcohols / Q000737:chemistry; D002417:Cattle; D002457:Cell Extracts / Q000032:analysis* / Q000737:chemistry; D004583:Electrons; D006146:Guanidines / Q000737:chemistry; D008264:Macrophages; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000737:chemistry; D055672:Static Electricity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001592:Benzyl Alcohols; D002457:Cell Extracts; D006146:Guanidines; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; C046886:3-nitrobenzyl alcohol