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

Abstract

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.

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…The SWATH-MS and the SWATH-like hyperreaction monitoring (HRM) techniques provide S/MRM-like performance in terms of reproducibility and quantitative accuracy and were implemented in a fast-scanning, high-resolution quadrupole time-of-flight and a quadrupole orbitrap mass spectrometer, respectively (Fig. 2, lower panel) (68,117). In SWATH or HRM mode, the instrument cycles repeatedly through fixed or variable adjacent precursor isolation windows (typically 32 windows of 25 m/z covering 400 -1200 m/z) over the course of chromatographic elution, thus fragmenting all coeluting precursor ions in each window recording multiplexed fragment ion spectra of all peptides in a userdefined retention time versus mass to charge window.…”

Applications of targeted proteomics in systems biology and translational medicine

“…Similar to SRM‐MS, PRM‐MS (parallel reaction monitoring mass spectrometry) takes advantage of a mass spectrometer which first selects precursors using a triple quadrupole and subsequently fragment ions are mass analyzed in an orbitrap 136. Using the same instrumentation as PRM‐MS, the instrument method can be adapted to allow for data independent acquisition 137, 138. Instead of coupling a quadrupole with an orbitrap mass analyzer, SWATH‐MS (sequential window acquisition of all theoretical masses) was implemented on a mass spectrometer which selects the precursors with a quadrupole and all fragment ions are analyzed by TOF 139.…”

Systematic evaluation of data‐independent acquisition for sensitive and reproducible proteomics‐a prototype design for a single injection assay

“…Furthermore, this finding is also relevant for recently described DIA methods that utilize varying isolation window widths based on precursor-ion densities that are not held constant across specific precursor m/z ranges and samples analyzed -because such assays rely on MS1 EICs for relative peptide quantification. [29] While the quantitative comparison of MS1 EICs compared to fragment EICs and QTOF instrument parameters in this study provides a framework for implementing DIA, future studies on other instruments such as quadrupole-Orbitraps are needed to ensure the findings are relevant across different mass spectrometers.…”