Simplifying sample handling for protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate-reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774.
Cordeiro
Francisco M
FM
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal.
Carreira
Ricardo J
RJ
Rial-Otero
Raquel
R
Rivas
M Gabriela
MG
Moura
Isabel
I
Capelo
Jose-Luis
JL
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002417:Cattle; D045424:Complex Mixtures / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D045424:Complex Mixtures; D010446:Peptide Fragments