on 03-06-201504:58 AM - edited on 10-15-202106:10 AM by AnalyteGuru
Bruderer R, Bernhardt OM, Gandhi T, Miladinovic SM, Cheng LY, Messner S, Ehrenberger T, Zanotelli V, Butscheid Y, Escher C, Vitek O, Rinner O, Reiter L. Mol Cell Proteomics. 2015 Feb 27. pii: mcp.M114.044305. The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared to the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention time normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements, and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98 % resulting in quasi complete data sets compared to 49 % of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP) treated 3D human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6 and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.