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.
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.
Carreira
R J
RJ
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal.
Cordeiro
F M
FM
Moro
A J
AJ
Rivas
M G
MG
Rial-Otero
R
R
Gaspar
E M
EM
Moura
I
I
Capelo
J L
JL
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
D011506:Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase