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Highly efficient enrichment and subsequent digestion of proteins in the mesoporous molecular sieve silicate SBA‐15 for matrix‐assisted laser desorption/ionization mass spectrometry with time‐of‐flight/time‐of‐flight analyzer peptide mapping
Author(s) -
Zuo Chao,
Yu Wenjia,
Zhou Xufeng,
Zhao Dongyuan,
Yang Pengyuan
Publication year - 2006
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2709
Subject(s) - chemistry , mass spectrometry , chromatography , myoglobin , desorption , sample preparation in mass spectrometry , time of flight mass spectrometry , mesoporous material , matrix assisted laser desorption/ionization , adsorption , analytical chemistry (journal) , electrospray ionization , biochemistry , ionization , ion , organic chemistry , catalysis
Based on a previous study of protein digestion inside the nanoreactor channels of the mesoporous molecular sieve silicate SBA‐15 ( Chem. Eur. J. 2005, 11: 5391), we have developed a highly efficient enrichment and subsequent tryptic digestion of proteins in SBA‐15 for matrix‐assisted laser desorption/ionization mass spectrometry with time‐of‐flight/time‐of‐flight analyzer (MALDI‐TOF/TOF) peptide mapping. The performance of the method is exemplified with myoglobin and cytochrome c. First, protein adsorption isotherms for two standard proteins with a range of initial concentration of proteins were investigated at room temperature. The results revealed that the kinetic adsorption rate of a protein within SBA‐15 was independent of initial protein concentration, and a 15‐min protein enrichment within SBA‐15 could be enough for protein identification in biological samples. It was noticed that no washing steps were needed to avoid protein loss due to desorption from the mesochannels into solution. Second, protein digestion inside the channels of SBA‐15 was also optimized. After adsorption of proteins into SBA‐15 in 15 min, the trypsin solution (pH 8) was directly added to the SBA‐15 beads with immobilized proteins by centrifugation, and then the digestion was performed for 15 min at 37°C. It was observed that a higher peptide sequence covering of 98% for myoglobin was obtained by MALDI‐TOF/TOF analysis, compared to in‐solution digestion. So the protein digestion inside SBA‐15 was proved to be significantly faster and yielded a better sequence coverage. The new procedure allows for rapid protein enrichment and digestion inside SBA‐15, and has great potential for protein analysis. Copyright © 2006 John Wiley & Sons, Ltd.