Research
Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*[S]

https://doi.org/10.1074/mcp.M114.044305Get rights and content
Under a Creative Commons license
open access

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.

Cited by (0)

Author contributions: R.B., S.M., Y.B., C.E., and L.R. designed the research; R.B., S.M., and V.Z. performed the research; R.B., O.M.B., T.G., S.M.M., L.C., T.E., and L.R. contributed new reagents or analytic tools; R.B., O.M.B., L.C., and L.R. analyzed data; R.B., S.M., O.V., O.R., and L.R. wrote the paper.

*

R.B. and S.M.M. acknowledge support from the Commission for Technology and Innovation, Switzerland, under the Grant No. 13539.1 PFFLI-LS.

Competing financial interests: The authors R.B., O.M.B., T.G., S.M.M., C.E., Y.B., O.R. and L.R. work for Biognosys AG.

4

Bernhardt, O. M., Selevsek, N., Gillet, L. C., Rinner, O., Picotti, P., Aebersold, R., and Reiter, L. (2012) Spectronaut A fast and efficient algorithm for MRM-like processing of data independent acquisition (SWATH-MS) data. Proceedings of the 60th ASMS Conference on Mass Spectrometry and Allied Topics, 2012, Vancouver, BC, Canada.

1

    The abbreviations used are:

    APAP

    acetaminophen

    ATP

    adenosine triphosphate

    CV

    coefficient of variation

    DIA

    data-independent acquisition

    DDA

    data-dependent acquisition

    FDR

    false discovery rate

    HRM

    hyper reaction monitoring

    iRT

    indexed retention time

    NAPQI

    N-acetyl-p-benzoquinone imine

    SRM

    selected reaction monitoring; DIA with 32 sequential windows of 25 Dalton width.

[S]

This article contains supplemental material files HRM-Spectral-libraries.xlsx, Profiling-Standard-Data-Set-table.xlsx, and Micro-tissue-Data-Set-tables.xlsx; Tables 1–5, and Figs. 1–9.

‡‡

These authors contributed to this work equally.