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

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

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.

MeSH terms

  • Acetaminophen / pharmacology*
  • Amidinotransferases / analysis
  • Amidinotransferases / genetics
  • Amidinotransferases / metabolism
  • Ammonia-Lyases / analysis
  • Ammonia-Lyases / genetics
  • Ammonia-Lyases / metabolism
  • Analgesics, Non-Narcotic / pharmacology*
  • Annexin A2 / analysis
  • Annexin A2 / genetics
  • Annexin A2 / metabolism
  • Gene Expression
  • Glutamate Formimidoyltransferase / analysis
  • Glutamate Formimidoyltransferase / genetics
  • Glutamate Formimidoyltransferase / metabolism
  • Hepatocytes / drug effects*
  • Hepatocytes / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / analysis
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Liver / drug effects*
  • Liver / metabolism
  • Oncogene Proteins / analysis
  • Oncogene Proteins / genetics
  • Oncogene Proteins / metabolism
  • Peptides / analysis*
  • Peroxiredoxin VI / analysis
  • Peroxiredoxin VI / genetics
  • Peroxiredoxin VI / metabolism
  • Protein Deglycase DJ-1
  • Proteolysis
  • Proteome / analysis*
  • Proteome / genetics
  • Proteome / metabolism
  • Proteomics / methods
  • Tissue Culture Techniques
  • Trypsin / chemistry
  • Voltage-Dependent Anion Channel 2 / analysis
  • Voltage-Dependent Anion Channel 2 / genetics
  • Voltage-Dependent Anion Channel 2 / metabolism

Substances

  • ANXA2 protein, human
  • Analgesics, Non-Narcotic
  • Annexin A2
  • Intracellular Signaling Peptides and Proteins
  • Oncogene Proteins
  • Peptides
  • Proteome
  • VDAC2 protein, human
  • Voltage-Dependent Anion Channel 2
  • Acetaminophen
  • PRDX6 protein, human
  • Peroxiredoxin VI
  • FTCD protein, human
  • Glutamate Formimidoyltransferase
  • Amidinotransferases
  • glycine amidinotransferase
  • PARK7 protein, human
  • Protein Deglycase DJ-1
  • Trypsin
  • Ammonia-Lyases