Label-free quantification in ion-mobility enhanced data-independent acquisition proteomics

ABSTRACT: Data-dependent acquisition (DDA) methods are a well-established tool for proteome analysis and have greatly expedited the field of shotgun proteomics. However, their serial and stochastic nature restricts their reproducibility and limits the detectable dynamic range to the peptides that ionize best. Unbiased data-independent acquisition (DIA) strategies strive to overcome this limitation and have gained increased popularity over recent years. The integration of ion-mobility separation (IMS) into DIA workflows provides an additional dimension of separation and 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 uses drift time specific collision energies to improve precursor fragmentation efficiency. The protocol comprises a detailed description of all major steps including sample preparation, LC-IMS-MS analysis, and data processing. Our protocol can handle proteome samples of any complexity and enables a highly reproducible and accurate label-free quantitation of up to 5,600 proteins across multiple runs in complete cellular lysates. Depending on the number of samples to be analysed, the protocol takes a minimum of three days to complete from proteolytic digest to data evaluation.

INSTRUMENT(S): Synapt G2-S HDMS

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Saccharomyces Cerevisiae (strain Lalvin Ec1118 / Prise De Mousse) (baker's Yeast)

TISSUE(S): Tissues

SUBMITTER: Ute Distler  

LAB HEAD: Stefan Tenzer

PROVIDER: PXD002393 | Pride | 2016-03-29

REPOSITORIES: Pride