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.Quantitative mass spectrometry is a powerful and widely used approach to identify differentially abundant proteins,
e.g. for proteome profiling and biomarker discovery (
1). Several tens of thousands of peptides and thousands of proteins can be routinely identified from a single sample injection in shotgun proteomics (
2). Shotgun proteomics, however, is limited by low analytical reproducibility. This is due to the complexity of the samples that results in under sampling (
supplemental Fig. 1) and to the fact that the acquisition of MS2 spectra is often triggered outside of the elution peak apex. As a result, only 17% of the detectable peptides are typically fragmented, and less than 60% of those are identified. This translates in reliable identification of only 10% of the detectable peptides (
3). The overlap of peptide identification across technical replicates is typically 35–60% (
4), which results in inconsistent peptide quantification. Alternatively to shotgun proteomics, selected reaction monitoring (SRM) enables quantification of up to 200–300 peptides at very high reproducibility, accuracy, and precision (
5–
8).Data-independent acquisition (DIA), a novel acquisition type, overcomes the semistochastic nature of shotgun proteomics (
9–
18). Spectra are acquired according to a predefined schema instead of dependent on the data. Targeted analysis of DIA data was introduced with SWATH-MS (
19). For the originally published SWATH-MS, the mass spectrometer cycles through 32 predefined, contiguous, 25 Thomson wide precursor windows, and records high-resolution fragment ion spectra (
19). This results in a comprehensive measurement of all detectable precursors of the selected mass range. The main novelty of SWATH-MS was in the analysis of the collected DIA data. Predefined fragment ions are extracted using precompiled spectrum libraries, which results in SRM-like data. Such targeted analyses are now enabled by several publicly available computational tools, in particular Spectronaut
2, Skyline (
20), and OpenSWATH (
21). The accuracy of peptide identification is evaluated based on the mProphet method (
22).We introduce a novel SWATH-MS-type DIA workflow termed hyper reaction monitoring (HRM) (reviewed in (
23)) implemented on a Thermo Scientific Q Exactive platform. It consists of comprehensive DIA acquisition and targeted data analysis with retention-time-normalized spectral libraries (
24). Its high accuracy of peptide identification and quantification is due to three aspects. First, we developed a novel, improved DIA method. Second, we reimplemented the mProphet (
22) approach in the software Spectronaut (
www.spectronaut.org). Third, we developed large, optimized, and retention-time-normalized (iRT) spectral libraries.We compared HRM and state-of-the-art shotgun proteomics in terms of ability to discover differentially abundant proteins. For this purpose, we used a “profiling standard sample set” with 12 non-human proteins spiked at known absolute concentrations into a stable human cell line protein extract. This resulted in quasi complete data sets for HRM and the detection of a larger number of differentially abundant proteins as compared with shotgun proteomics. We utilized HRM to identify changes in the proteome in primary three-dimensional human liver microtissues after APAP exposure (
25–
27). These primary hepatocytes exhibit active drug metabolism. With a starting material of only 12,000 cells per sample, the abundance of 2,830 proteins was quantified over an APAP concentration range. Six novel NAPQI-cysteine proteins adducts that might be relevant for the toxicity of APAP were found and quantified mainly on mitochondrion-related proteins.
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