title: Exploring skyline for both MS(E) -based label-free proteomics and HRMS quantitation of small molecules.
abstract: The MS(E) (where MS(E) is low energy (MS) and elevated energy (E) mode of acquisition) acquisition method commercialized by Waters on its Q-TOF instruments is regarded as a unique data-independent fragmentation approach that improves the accuracy and dynamic range of label-free proteomic quantitation. Due to its special format, MS(E) acquisition files cannot be independently analyzed with most widely used open-source proteomic software specialized for processing data-dependent acquisition files. In this study, we established a workflow integrating Skyline, a popular and versatile peptide-centric quantitation program, and a statistical tool DiffProt to fulfill MS(E) -based proteomic quantitation. Comparison with the vendor software package for analyzing targeted phosphopeptides and global proteomic datasets reveals distinct advantages of Skyline in MS(E) data mining, including sensitive peak detection, flexible peptide filtering, and transparent step-by-step workflow. Moreover, we developed a new procedure such that Skyline MS1 filtering was extended to small molecule quantitation for the first time. This new utility of Skyline was examined in a protein-ligand interaction experiment to identify multiple chemical compounds specifically bound to NDM-1 (where NDM is New Delhi metallo-β-lactamase 1), an antibiotics-resistance target. Further improvement of the current weaknesses in Skyline MS1 filtering is expected to enhance the reliability of this powerful program in full scan-based quantitation of both peptides and small molecules.
authors.affiliation: College of Life Sciences, Nankai University, Tianjin, P. R. China; High-Throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin, P. R. China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Liu
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Yan
authors.forename: Shanshan
authors.initials: S
authors.lastname: Qin
authors.forename: Jinhua
authors.initials: J
authors.lastname: Xu
authors.forename: Jianping
authors.initials: J
authors.lastname: Lin
authors.forename: Cheng
authors.initials: C
authors.lastname: Yang
authors.forename: Wenqing
authors.initials: W
authors.lastname: Shui
mesh_terms: D000595:Amino Acid Sequence; D057225:Data Mining; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis; D011485:Protein Binding; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012984:Software; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D010748:Phosphopeptides; D020543:Proteome; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1
keywords: Bioinformatics; MS1 filtering; MSE; Proteomic quantitation; Skyline; Small molecule quantitation