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Title of article :
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
Author/Authors :
Carreira، نويسنده , , R.J. and Cordeiro، نويسنده , , F.M. and Moro، نويسنده , , A.J. and Rivas، نويسنده , , M.G. and Rial-Otero، نويسنده , , R. and Gaspar، نويسنده , , E.M. and Moura، نويسنده , , I. and Capelo، نويسنده , , J.L.، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2007
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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 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.
Keywords :
Protein digestion , MALDI-TOF-MS , High-intensity focused ultrasound
Journal title :
Journal of Chromatography A
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