High precision iRT retention time prediction in the targeted analysis of data-independent acquisition and its impact on identification and quantitation

ABSTRACT: Targeted analysis of DIA data is a powerful mass spectrometric approach for comprehensive, reproducible and precise proteome quantitation. It requires a spectral library, which contains for all considered peptide precursor ions empirically determined fragment ion intensities and their predicted retention time. Retention times, however, are not comparable on an absolute scale, especially if heterogeneous measurements are combined. Here we present a method for high-precision prediction of retention time, which significantly improves the quality of targeted DIA analysis compared to in silico retention time prediction and the state of the art indexed retention time (iRT) normalization approach. We describe a high precision normalized retention time algorithm, which is implemented in the Spectronaut software. We furthermore investigate the influence of nine different experimental factors, such as chromatographic mobile and stationary phase, on iRT precision. In summary we show that using targeted analysis of DIA data with high precision iRT significantly increases sensitivity and data quality. The iRT values are generally transferable across a wide range of experimental conditions. Best results, however, are achieved if library generation and analytical measurements are performed on the same system.

INSTRUMENT(S): TripleTOF 5600, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Roland Bruderer  

LAB HEAD: Lukas Reiter

PROVIDER: PXD004188 | Pride | 2016-07-14

REPOSITORIES: Pride