Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.
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.
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland; §Institute of Molecular Systems Biology, ETH Zurich, Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland;
Roland
R
Bruderer
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Oliver M
OM
Bernhardt
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Tejas
T
Gandhi
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland; §Institute of Molecular Systems Biology, ETH Zurich, Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland;
Saša M
SM
Miladinović
¶Department of Statistics, Department of Computer Science, Purdue University, 150 N. University Street, West Lafayette, IN 47907-2068;
Lin-Yang
LY
Cheng
**InSphero AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
Simon
S
Messner
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Tobias
T
Ehrenberger
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Vito
V
Zanotelli
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Yulia
Y
Butscheid
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Claudia
C
Escher
¶Department of Statistics, Department of Computer Science, Purdue University, 150 N. University Street, West Lafayette, IN 47907-2068;
Olga
O
Vitek
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland;
Oliver
O
Rinner
From the ‡Biognosys, Wagistrasse 25, CH-8952 Schlieren, Switzerland; reiter@biognosys.ch.
Lukas
L
Reiter
D000082:Acetaminophen / Q000494:pharmacology*; D000579:Amidinotransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000642:Ammonia-Lyases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D017306:Annexin A2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D051042:Glutamate Formimidoyltransferase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D064251:Multifunctional Enzymes; D015513:Oncogene Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000032:analysis*; D054465:Peroxiredoxin VI / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000071617:Protein Deglycase DJ-1; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D046509:Tissue Culture Techniques; D014357:Trypsin / Q000737:chemistry; D050996:Voltage-Dependent Anion Channel 2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism
C472822:ANXA2 protein, human; D018712:Analgesics, Non-Narcotic; D017306:Annexin A2; D047908:Intracellular Signaling Peptides and Proteins; D064251:Multifunctional Enzymes; D015513:Oncogene Proteins; D010455:Peptides; D020543:Proteome; C494765:VDAC2 protein, human; D050996:Voltage-Dependent Anion Channel 2; D000082:Acetaminophen; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; C494848:FTCD protein, human; D051042:Glutamate Formimidoyltransferase; D000579:Amidinotransferases; C020681:glycine amidinotransferase; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1; D014357:Trypsin; D000642:Ammonia-Lyases