Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies

John E. Wiktorowicz, Susan Stafford, Harriet Rea, Petri Urvil, Kizhake Soman, Alexander Kurosky, J. Regino Perez-Polo, Tor C. Savidge

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

Original languageEnglish (US)
Pages (from-to)5601-5614
Number of pages14
JournalBiochemistry
Volume50
Issue number25
DOIs
StatePublished - Jun 28 2011

Fingerprint

Proteomics
Fluorescence
Protein S
Electrophoretic mobility
S-Nitrosoglutathione
Neurons
Proteins
Calcium Signaling
Neurogenesis
Neurites
Post Translational Protein Processing
Reperfusion Injury
Morphogenesis
Neuroglia
Functional analysis
Nervous System
Reperfusion
Cysteine
Axons
Neurology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Wiktorowicz, J. E., Stafford, S., Rea, H., Urvil, P., Soman, K., Kurosky, A., ... Savidge, T. C. (2011). Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies. Biochemistry, 50(25), 5601-5614. https://doi.org/10.1021/bi200008b

Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies. / Wiktorowicz, John E.; Stafford, Susan; Rea, Harriet; Urvil, Petri; Soman, Kizhake; Kurosky, Alexander; Perez-Polo, J. Regino; Savidge, Tor C.

In: Biochemistry, Vol. 50, No. 25, 28.06.2011, p. 5601-5614.

Research output: Contribution to journalArticle

Wiktorowicz, JE, Stafford, S, Rea, H, Urvil, P, Soman, K, Kurosky, A, Perez-Polo, JR & Savidge, TC 2011, 'Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies', Biochemistry, vol. 50, no. 25, pp. 5601-5614. https://doi.org/10.1021/bi200008b
Wiktorowicz JE, Stafford S, Rea H, Urvil P, Soman K, Kurosky A et al. Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies. Biochemistry. 2011 Jun 28;50(25):5601-5614. https://doi.org/10.1021/bi200008b
Wiktorowicz, John E. ; Stafford, Susan ; Rea, Harriet ; Urvil, Petri ; Soman, Kizhake ; Kurosky, Alexander ; Perez-Polo, J. Regino ; Savidge, Tor C. / Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies. In: Biochemistry. 2011 ; Vol. 50, No. 25. pp. 5601-5614.
@article{19737c6779c54b408eb6048ff5747432,
title = "Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies",
abstract = "Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.",
author = "Wiktorowicz, {John E.} and Susan Stafford and Harriet Rea and Petri Urvil and Kizhake Soman and Alexander Kurosky and Perez-Polo, {J. Regino} and Savidge, {Tor C.}",
year = "2011",
month = "6",
day = "28",
doi = "10.1021/bi200008b",
language = "English (US)",
volume = "50",
pages = "5601--5614",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "25",

}

TY - JOUR

T1 - Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies

AU - Wiktorowicz, John E.

AU - Stafford, Susan

AU - Rea, Harriet

AU - Urvil, Petri

AU - Soman, Kizhake

AU - Kurosky, Alexander

AU - Perez-Polo, J. Regino

AU - Savidge, Tor C.

PY - 2011/6/28

Y1 - 2011/6/28

N2 - Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

AB - Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

UR - http://www.scopus.com/inward/record.url?scp=79959423082&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79959423082&partnerID=8YFLogxK

U2 - 10.1021/bi200008b

DO - 10.1021/bi200008b

M3 - Article

C2 - 21615140

AN - SCOPUS:79959423082

VL - 50

SP - 5601

EP - 5614

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 25

ER -