RT info:eu-repo/semantics/article
T1 Updates of the In¿Gel Digestion Method for Protein Analysis by Mass Spectrometry
A1 Hernández Fernaud, Juan Ramón
A1 Goodman, Jennifer K.
A1 Zampronio, Cleidiane G.
A1 Jones, Alexandra M. E.
A2 BioquímicaMicrobiología, Biología Celular y Genética
A2 School of Life Sciences. University of Warwick.
AB The in-gel digestion of proteins for analysis by liquid chromatograph massspectrometry has been used since the early 1990s. Although severalimprovements have contributed to increasing the quality of the data obtained,many recent publications still use sub-optimal approaches. Updates of thein-gel digestion protocol has been presented in the study. It has been shownthat alternative reducing, alkylating agent reactions, and tryptic digestionbuffers increase peptide and protein identification and reduce incubationtimes. The results indicate that a simultaneous and short, high temperaturereduction and alkylation reaction using Tris(2-carboxyethyl)phosphinehydrochloride and chloroacetamide with a subsequent gel wash improveprotein identification and sequence coverage, and diminish peptide sidereactions. Additionally, use of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonicacid buffer allows a significant reduction in the digestion time improvingtrypsin performance and increasing the peptide recovery. The updates of thein-gel digestion protocol described here are efficient and offer flexibility to beincorporated in any proteomic laboratory.
YR 2018
FD 2018
LK http://riull.ull.es/xmlui/handle/915/38838
UL http://riull.ull.es/xmlui/handle/915/38838
LA en
NO DOI: 10.1002/pmic.201800236
DS Repositorio institucional de la Universidad de La Laguna
RD 05-ene-2025