Application of the thiosulfonate switch technique and a modified biotin switch technique protocols to detect protein S-nitrosothiols in mouse liver lysates in 1D and 2D gel studies

dc.contributor.advisorChairperson, Graduate Committee: Paul Grieco; Brian Bothner (co-chair)en
dc.contributor.authorMiller, Colin Gregoryen
dc.date.accessioned2017-02-02T17:40:58Z
dc.date.available2017-02-02T17:40:58Z
dc.date.issued2015en
dc.description.abstractWhile the role of nitric oxide (NO) in cell signaling and liver growth has been well documented, the identification of S-nitrosylated proteins, one of the major NO transport mechanisms within the cell, remains a challenge. Classically, the implementation of biotin labeling, known as the biotin switch technique (BST), with streptavidin-agarose bead pulldown and subsequent immunoblotting, has offered the best results for identifying S-nitrosocysteine residues within proteins. However, this technique has come under scrutiny for its use of ascorbate as a reducing agent. Numerous published accounts have shown ascorbate's poor reducing potential especially for S-nitrosoproteins. To this end, the Grieco lab has shown that pure S-nitrosylated proteins can be transformed into S-phenylsulfonylcysteine residues, which can be readily converted into mixed disulfides thus allowing for labeling of pure nitrosylated proteins at pH's as low as pH 4. This protocol is referred to as the thiosulfonate switch technique (TST). The Grieco lab has also modified the biotin switch technique to incorporate electrophilic maleimide and orthopyridyl disulfide (OPSS) dyes for fluorescence labeling of the S-nitroso proteome. To examine the scope and limitations of the TST vis-a-vis cell lysates, the TST protocol and the modified BST protocol, both employing novel Z-CyDyes developed in the Grieco Laboratory, have been used to specifically label S-nitrosylated proteins in complex liver lysates. The successful labeling of mouse liver lysates, employing UV and ascorbate SNO knockout negative controls, is demonstrated in both 1D and 2D gel studies. Also reported herein is the creation of novel second generation maleimide dyes (SO3-Cy-Mal) based on Z-CyDyes.en
dc.identifier.urihttps://scholarworks.montana.edu/handle/1/10159en
dc.language.isoenen
dc.publisherMontana State University - Bozeman, College of Letters & Scienceen
dc.rights.holderCopyright 2015 by Colin Gregory Milleren
dc.subject.lcshThiolsen
dc.subject.lcshProteomicsen
dc.subject.lcshAmino acidsen
dc.titleApplication of the thiosulfonate switch technique and a modified biotin switch technique protocols to detect protein S-nitrosothiols in mouse liver lysates in 1D and 2D gel studiesen
dc.typeThesisen
mus.data.thumbpage40en
thesis.degree.committeemembersMembers, Graduate Committee: Edward E. Schmidt; Mary J. Cloninger.en
thesis.degree.departmentChemistry & Biochemistry.en
thesis.degree.genreThesisen
thesis.degree.nameMSen
thesis.format.extentfirstpage1en
thesis.format.extentlastpage72en

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