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

• Published in: Journal of Chromatography A Vol. 1153; no. 1; pp. 291 - 299
• Main Authors: Carreira, R.J., Cordeiro, F.M., Moro, A.J., Rivas, M.G., Rial-Otero, R., Gaspar, E.M., Moura, I., Capelo, J.L.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.06.2007
• Subjects: Amino Acid Sequence; Animals; Desulfovibrio desulfuricans - enzymology; Electrophoresis, Polyacrylamide Gel - methods; High-intensity focused ultrasound; Humans; MALDI-TOF-MS; Molecular Sequence Data; Oxidoreductases Acting on Sulfur Group Donors - isolation & purification; Protein digestion; Proteins - analysis; Proteins - chemistry; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Ultrasonics; Protein digestion; MALDI-TOF-MS; High-intensity focused ultrasound
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2006.09.078

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 μL. 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 μg. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 μg/mL.