Selective fluorescent labeling of S-nitrosothiols (S-FLOS): a novel method for studying S-nitrosation.
Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.
Santhanam
Lakshmi
L
The Johns Hopkins School of Medicine, Anesthesiology and CCM, 720 Rutland Avenue, Traylor 621, Baltimore, MD 21205, USA.
Gucek
Marjan
M
Brown
Tashalee R
TR
Mansharamani
Malini
M
Ryoo
Sungwoo
S
Lemmon
Christopher A
CA
Romer
Lewis
L
Shoukas
Artin A
AA
Berkowitz
Dan E
DE
Cole
Robert N
RN
D000818:Animals; D001710:Biotin; D001923:Brain Chemistry; D002232:Carbocyanines; D030562:Databases, Protein; D005453:Fluorescence; D008301:Maleimides; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D015538:Nitrosation; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D026403:S-Nitrosothiols / Q000032:analysis; D016622:Silver Staining; D053719:Tandem Mass Spectrometry
D002232:Carbocyanines; D008301:Maleimides; D011506:Proteins; D026403:S-Nitrosothiols; C089187:cyanine dye 3; C085321:cyanine dye 5; C043592:maleimide; D009569:Nitric Oxide; D001710:Biotin; D019001:Nitric Oxide Synthase