title: New findings for in-gel digestion accelerated by high-intensity focused ultrasound for protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
abstract: New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 microL. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 microg. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 microg/mL.
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal.
authors.forename: R J
authors.initials: RJ
authors.lastname: Carreira
authors.forename: F M
authors.initials: FM
authors.lastname: Cordeiro
authors.forename: A J
authors.initials: AJ
authors.lastname: Moro
authors.forename: M G
authors.initials: MG
authors.lastname: Rivas
authors.forename: R
authors.initials: R
authors.lastname: Rial-Otero
authors.forename: E M
authors.initials: EM
authors.lastname: Gaspar
authors.forename: I
authors.initials: I
authors.lastname: Moura
authors.forename: J L
authors.initials: JL
authors.lastname: Capelo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045285:Desulfovibrio desulfuricans / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014465:Ultrasonics
chemical_list: D011506:Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase