6533b871fe1ef96bd12d109b
RESEARCH PRODUCT
Label-free quantification in ion mobility-enhanced data-independent acquisition proteomics.
Jörg KuharevUte DistlerStefan TenzerPedro Navarrosubject
0301 basic medicineProteomicsTime FactorsProteomeComputer scienceQuantitative proteomicsProteolytic enzymesProteinsProteomicsMass spectrometryGeneral Biochemistry Genetics and Molecular BiologyChemistry Techniques AnalyticalMass Spectrometry03 medical and health sciencesLabel-free quantification030104 developmental biologyProteomeHumansData-independent acquisitionSample preparationBiological systemChromatography LiquidHeLa Cellsdescription
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.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2016-03-25 | Nature protocols |