Abstract
High-resolution two-dimensional gel electrophoresis and in-gel digestion are routinely used for large-scale protein separation and peptide generation in mass spectrometry-based proteomics, respectively. However, the combination of isoelectric focusing in the first dimension and polyacrylamide slab gel electrophoresis in the second dimension is not suitable for the proper separation of integral proteins and high-molecular-mass proteins. In addition, in-gel trypsination may not result in a high degree of efficient digestion levels for the production of large numbers of peptides in the case of certain protein species. The application of gradient one-dimensional gel electrophoresis and on-membrane digestion can overcome these technical problems and be extremely helpful for the comprehensive identification of proteins that are underrepresented in routine two-dimensional gel electrophoretic approaches. This review critically examines the general application of on-membrane digestion techniques in proteomics and its recent application for the identification of very large integral membrane proteins from skeletal muscle by mass spectrometry. This includes the discussion of proteomic studies that have focused on the proteomic characterization of the membrane cytoskeletal protein dystrophin from sarcolemma vesicles and the ryanodine receptor calcium release channel of the sarcoplasmic reticulum from skeletal muscle.
Highlights
The usage of adsorbent membranes as support material for blotted biomolecules is widely used in analytical biochemistry [1]
In some cases trypsination is inefficient for comprehensive in-gel digestion and alternative techniques with membrane replicas of gels have been shown to result in superior results
The recent application of on-membrane digestion in skeletal muscle proteomics has resulted in the mass spectrometric identification of extremely large membrane-associated proteins, i.e. the Dp427 isoform of dystrophin and the RyR1 isoform of the ryanodine receptor Ca2+-release channel
Summary
The usage of adsorbent membranes as support material for blotted biomolecules is widely used in analytical biochemistry [1]. The efficient transfer of DNA, RNA or protein from a gel system onto a membrane support is critical in these frequently used techniques [5, 6]. In-gel trypsination is the most frequently used method to generate peptides from gel electrophoretically separated protein mixtures [34, 35]. This technique does sometimes not result in an efficient proteolysis of distinct protein species for their subsequent identification by mass spectrometry, which necessitates the application of alternative methods such as on-membrane trypsination [21, 22].
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