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

被引:32
作者
Carreira, R. J. [1 ]
Cordeiro, F. M. [1 ]
Moro, A. J. [1 ]
Rivas, M. G. [1 ]
Rial-Otero, R. [1 ]
Gaspar, E. M. [1 ]
Moura, I. [1 ]
Capelo, J. L. [1 ]
机构
[1] Univ Nova Lisboa, Fac Ciencias & Tecnol, Dept Quim, REQUIMTE, P-2829516 Monte De Caparica, Portugal
关键词
protein digestion; high-intensity focused ultrasound; MALDI-TOF-MS;
D O I
10.1016/j.chroma.2006.09.078
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 mu L. 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 mu g. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 mu g/mL. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:291 / 299
页数:9
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