Send to

Choose Destination
Mol Cell Proteomics. 2015 May;14(5):1400-10. doi: 10.1074/mcp.M114.044305. Epub 2015 Feb 27.

Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.

Author information

From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland; §Institute of Molecular Systems Biology, ETH Zurich, Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland;
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
¶Department of Statistics, Department of Computer Science, Purdue University, 150 N. University Street, West Lafayette, IN 47907-2068;
**InSphero AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;


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 with 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 with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional 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.

[Indexed for MEDLINE]
Free PMC Article

MeSH terms, Substances

MeSH terms


Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center