Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

PASS-DIA: A novel data-independent acquisition approach for discovery studies (#127)

Akhilesh Pandey 1 2 3 4 5 , Dong-Gi Mun 1 2 , Santosh Renuse 1 2 , Mayank Saraswat 2 3 4 5 , Anil Madugundu 1 2 3 , Savita Udainiya 1 2 , Hui Zhao 2 , Raja Sekhar Nirujogi 4 6 , Chan-Hyun Na 6 7 , Nagarajan Kannan 2 , Sang-Won Lee 8
  1. Center for Individualized Medicine, Mayo Clinic, Rochester, USA
  2. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, USA
  3. Center for Molecular Medicine, National Institute of Mental Health and Neurosciences , Bangalore, India
  4. Institute of Bioinformatics, International Technology Park, Bangalore, India
  5. Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India
  6. McKusick-Nathans Institute of Genetic Medicine , Johns Hopkins University School of Medicine, Baltimore, USA
  7. Neurology, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, USA
  8. Department of Chemistry, Center for Proteogenome Research, Korea University, Seoul, Republic of Korea

Data-independent acquisition (DIA) approach is being increasingly adopted as a promising strategy for identification and quantitation of known peptides across a large set of samples. As most of DIA datasets are acquired with much wider isolation windows than data-dependent acquisition (DDA) experiments, complex MS/MS spectra are generated, which hampers obtaining maximum peptide information through classical protein database search methods. Therefore, analysis of DIA data mainly relies on evidence of existence of peptides from pre-built spectral libraries using a peptide-centric approach. Consequently, one major weakness of this method is that it does not account for peptides which are not included in spectral library, precluding the use of DIA for discovery studies. Here, we present a strategy termed PASS-DIA (Precursor ion And Small Slice-DIA) in which MS/MS spectra are acquired with small isolation windows and MS/MS spectra are interpreted with accurately measured precursor ion masses. This method enables direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was superior to both DDA and conventional DIA experiments with regard to identification of peptides. Application of PASS-DIA for analysis of samples with post-translationally modified peptides such as phosphorylation and N-glycosylation again revealed its superior performance. Finally, the use of PASS-DIA to characterize a rare proteome of human fallopian tube organoid samples revealed biologically relevant and low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool which outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that PASS-DIA method will become an important strategy for discovery type studies when deeper proteome characterization is necessitated.