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

[ Vol. 19 , Issue. 3 ]

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.



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