title: Label-free quantification in ion mobility-enhanced data-independent acquisition proteomics.
abstract: Unbiased data-independent acquisition (DIA) strategies have gained increased popularity in the field of quantitative proteomics. The integration of ion mobility separation (IMS) into DIA workflows provides an additional dimension of separation to liquid chromatography-mass spectrometry (LC-MS), and it increases the achievable analytical depth of DIA approaches. Here we provide a detailed protocol for a label-free quantitative proteomics workflow based on ion mobility-enhanced DIA, which synchronizes precursor ion drift times with collision energies to improve precursor fragmentation efficiency. The protocol comprises a detailed description of all major steps including instrument setup, filter-aided sample preparation, LC-IMS-MS analysis and data processing. Our protocol can handle proteome samples of any complexity, and it enables a highly reproducible and accurate precursor intensity-based label-free quantification of up to 5,600 proteins across multiple runs in complete cellular lysates. Depending on the number of samples to be analyzed, the protocol takes a minimum of 3 d to complete from proteolytic digestion to data evaluation.
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany.|Focus Program Translational Neuroscience (FTN), University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Distler
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany.
authors.forename: Jörg
authors.identifier: http://orcid.org/0000-0001-9722-9474
authors.initials: J
authors.lastname: Kuharev
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany.
authors.forename: Pedro
authors.identifier: http://orcid.org/0000-0002-9052-1824
authors.initials: P
authors.lastname: Navarro
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany.
authors.forename: Stefan
authors.identifier: http://orcid.org/0000-0003-3034-0017
authors.initials: S
authors.lastname: Tenzer
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
chemical_list: D011506:Proteins; D020543:Proteome