期刊
PROTEIN AND PEPTIDE LETTERS
卷 19, 期 3, 页码 252-263出版社
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/092986612799363208
关键词
Calcium homeostasis; mass spectrometry; muscle proteomics; on-membrane digestion; ryanodine receptor; sarcoplasmic reticulum
资金
- Irish Health Research Board [HRB-RP/2008/1]
- Higher Education Authority [HEA-RERGS-07-NUIM]
- Health Research Board [HRB-EQ/2003/3, HRB-EQ/2004/2]
The sarcoplasmic reticulum from skeletal muscle constitutes an elaborate membrane system that contains a considerable number of integral and very large proteins that exist in highly complex supramolecular clusters. Conventional proteomics using two-dimensional gel electrophoresis greatly underestimates the presence of these proteins. Here, we have applied one-dimensional gradient gels and on-membrane digestion to overcome this technical problem. Mass spectrometric analysis has determined the presence of 31 distinct protein species in the sarcoplasmic reticulum, including key Ca2+-handling proteins such as the ryanodine receptor, Ca2+-ATPase, calsequestrin and sarcalumenin. Immunoblotting confirmed the relative position of these Ca2+-regulatory elements in analytical gel replicas. Interestingly, aldolase and phosphofructokinase were found to be present in the purified sarcoplasmic reticulum, supporting the idea of a close physical coupling between the glycolytic pathway and the energy-dependent sarcoplasmic reticulum. Hence, on-membrane digestion is highly suitable as the method of choice for studying integral and high-molecular-mass proteins in proteomic studies.
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