Proteoform-specific insights into cellular proteome regulation

Emma L. Norris, Madeleine J. Headlam, Keyur A. Dave, David D. Smith, Alexander Bukreyev, Toshna Singh, Buddhika A. Jayakody, Keith J. Chappell, Peter L. Collins, Jeffrey J. Gorman

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)3297-3320
Number of pages24
JournalMolecular and Cellular Proteomics
Volume15
Issue number10
DOIs
StatePublished - Oct 1 2016
Externally publishedYes

Fingerprint

Human respiratory syncytial virus
Proteome
Viruses
Proteins
Alternative Splicing
Proteolysis
Phosphorylation
Template matching
Mass Spectrometry
Biological systems
Western Blotting
Mass spectrometry
Phenotype
Infection
Chemical analysis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Norris, E. L., Headlam, M. J., Dave, K. A., Smith, D. D., Bukreyev, A., Singh, T., ... Gorman, J. J. (2016). Proteoform-specific insights into cellular proteome regulation. Molecular and Cellular Proteomics, 15(10), 3297-3320. https://doi.org/10.1074/mcp.O116.058438

Proteoform-specific insights into cellular proteome regulation. / Norris, Emma L.; Headlam, Madeleine J.; Dave, Keyur A.; Smith, David D.; Bukreyev, Alexander; Singh, Toshna; Jayakody, Buddhika A.; Chappell, Keith J.; Collins, Peter L.; Gorman, Jeffrey J.

In: Molecular and Cellular Proteomics, Vol. 15, No. 10, 01.10.2016, p. 3297-3320.

Research output: Contribution to journalArticle

Norris, EL, Headlam, MJ, Dave, KA, Smith, DD, Bukreyev, A, Singh, T, Jayakody, BA, Chappell, KJ, Collins, PL & Gorman, JJ 2016, 'Proteoform-specific insights into cellular proteome regulation', Molecular and Cellular Proteomics, vol. 15, no. 10, pp. 3297-3320. https://doi.org/10.1074/mcp.O116.058438
Norris, Emma L. ; Headlam, Madeleine J. ; Dave, Keyur A. ; Smith, David D. ; Bukreyev, Alexander ; Singh, Toshna ; Jayakody, Buddhika A. ; Chappell, Keith J. ; Collins, Peter L. ; Gorman, Jeffrey J. / Proteoform-specific insights into cellular proteome regulation. In: Molecular and Cellular Proteomics. 2016 ; Vol. 15, No. 10. pp. 3297-3320.
@article{0e3fe290d6a640eb9c3c02b3a322afaf,
title = "Proteoform-specific insights into cellular proteome regulation",
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.",
author = "Norris, {Emma L.} and Headlam, {Madeleine J.} and Dave, {Keyur A.} and Smith, {David D.} and Alexander Bukreyev and Toshna Singh and Jayakody, {Buddhika A.} and Chappell, {Keith J.} and Collins, {Peter L.} and Gorman, {Jeffrey J.}",
year = "2016",
month = "10",
day = "1",
doi = "10.1074/mcp.O116.058438",
language = "English (US)",
volume = "15",
pages = "3297--3320",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "10",

}

TY - JOUR

T1 - Proteoform-specific insights into cellular proteome regulation

AU - Norris, Emma L.

AU - Headlam, Madeleine J.

AU - Dave, Keyur A.

AU - Smith, David D.

AU - Bukreyev, Alexander

AU - Singh, Toshna

AU - Jayakody, Buddhika A.

AU - Chappell, Keith J.

AU - Collins, Peter L.

AU - Gorman, Jeffrey J.

PY - 2016/10/1

Y1 - 2016/10/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84992163270&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84992163270&partnerID=8YFLogxK

U2 - 10.1074/mcp.O116.058438

DO - 10.1074/mcp.O116.058438

M3 - Article

VL - 15

SP - 3297

EP - 3320

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 10

ER -