Abstract
Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.
Highlights
From the ‡Protein Discovery Centre and §Statistics Unit, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia, ¶Respiratory Virus Section, Laboratory of Infectious Diseases, National Institute for Allergy and Infectious Diseases, NIH, Bethesda, Maryland, and ʈSchool of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia
The global analysis identified protein groups and cellular pathways affected by Human respiratory syncytial virus (hRSV) infection, insight into regulation of specific proteoforms was not apparent in this global approach
0.96 a Numbers in parentheses represent the portion of the full length UniProt sequence entry represented by the proteoform sequence. b Proteoform concordance values (⌬ ϭ 1) for the five uninfected experiments only (Mock), the five hRSV-infected experiments only and the ten IEF experiments combined (ALL). c Indicates whether the N-terminal/C-terminal peptide was identified in a fraction within the predicted focusing window. d N-terminal peptide arising from processing of the canonical sequence was not in the search space
Summary
A strategy is described for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. Correlation between experimental and theoretical pI gradients indicated reproducible focusing Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. E.L.N. conceived of and performed the computational and visualization methods for proteoform profiling and provided substantial authorship. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.
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