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Protein & Peptide Letters
ISSN (Print): 0929-8665
ISSN (Online): 1875-5305
VOLUME: 19
ISSUE: 3
DOI: 10.2174/092986612799363208      Price:  $58









Mass Spectrometric Characterization of the Sarcoplasmic Reticulum from Rabbit Skeletal Muscle by On-membrane Digestion

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Author(s): Lisa Staunton and Kay Ohlendieck
Pages 252-263 (12)
Abstract:
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.
Keywords:
Calcium homeostasis, mass spectrometry, muscle proteomics, on-membrane digestion, ryanodine receptor, sarcoplasmic reticulum, sarcalumenin, calsequestrin, gel electrophoresis
Affiliation:
Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.