title: Study of primary amines for nucleophilic cleavage of cyanylated cystinyl proteins in disulfide mass mapping methodology.
abstract: In a study of primary (methyl to butyl) amines as nucleophiles for cyano-induced cleavage of cysteinyl proteins, methylamine was found to be superior to ammonia for cyanylation (CN)-based disulfide mass mapping methodology. Reaction conditions such as nucleophile concentration, temperature, and reaction time were systematically studied using ribonuclease A as a model protein. The CN-induced cleavage products were monitored using reverse-phase chromatography and matrix-assisted laser desorption ionization mass spectrometry. Results showed that low temperature, short reaction time, and high nucleophile concentration optimize the cleavage reaction and minimize side reactions. These conditions shorten the analysis time and substantially improve the yield of cleavage products. Further, the concurrent use of homologous nucleophiles (e.g., ammonia and methylamine) facilitates recognition and identification of cleavage products.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: José-Luis
authors.initials: JL
authors.lastname: Gallegos-Pérez
authors.forename: Laura
authors.initials: L
authors.lastname: Rangel-Ordóñez
authors.forename: Stephen Robert
authors.initials: SR
authors.lastname: Bowman
authors.forename: Charles O
authors.initials: CO
authors.lastname: Ngowe
authors.forename: J Throck
authors.initials: JT
authors.lastname: Watson
mesh_terms: D000588:Amines / Q000737:chemistry*; D002082:Butylamines / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005021:Ethylamines / Q000737:chemistry; D007768:Lactalbumin / Q000737:chemistry; D008744:Methylamines / Q000737:chemistry; D011437:Propylamines / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D000588:Amines; D002082:Butylamines; D003486:Cyanides; D004220:Disulfides; D005021:Ethylamines; D008744:Methylamines; D011437:Propylamines; D020033:Protein Isoforms; D003553:Cystine; D007768:Lactalbumin; C027451:methylamine; D012259:Ribonuclease, Pancreatic; C041564:ethylamine