Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

Abstract

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

CD8 T-cell Recruitment Into the Central Nervous System of Cuprizone-Fed Mice: Relevance to Modeling the Etiology of Multiple Sclerosis

“…Briefly, mice (n = 3 animals/group) were euthanized and immediately thereafter brains, spinal cords, thymic and spleens were harvested, washed thoroughly with ice-cold 0.01 M phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (4 µM staurosporine, 1 mM α-naphthyl phosphate and 1 mM sodium orthovanadate) to prevent protein degradation during sample preparation and then snap-frozen in liquid nitrogen. Frozen samples were pulverized, solubilized (∼1 µl/1 µg tissue) in pre-chilled lysis buffer (25 mM Tris, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl and 1% Triton x-100) and centrifuged at 125,000 g, 4 • C, for 1 h. Total protein in the supernatant was quantified in each sample using the EZQ protein quantitation kit (Life Technologies, Eugene, OR, USA) with bovine serum albumin (Amresco, Solon, OH, USA) as a calibration standard (Butt and Coorssen, 2005;Churchward et al, 2005). Thymus, spleen, brain, spinal cord and standard purified CD4/8 recombinant proteins (Sino Biological, Wayne, PA, USA) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 100 V for 2 h at 4 • C) and then transferred onto polyvinylidene difluoride membrane (PVDF, 0.22 µM pore size, Bio-Rad, Hercules, CA, USA) for 2 h at 4 • C; transfer efficiency, determined as previously described (Sen et al, 2019a), was 95.6 ± 1.7% (Supplementary Figures S1A,B).…”

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

“…A comprehensive analysis of the Hela cell proteome by SILAC 2DE‐LC‐LTQ OrbiTrap XL revealed that 676 spots from 816 analyzed ones contained at least two with up to 22 different proteins . Combined with 2DE gel image analysis, conventional 2DE‐based comparative proteomics has been extensively used to determine “differentially expressed proteins” under different physiological, pathological, or pathophysiological conditions as reviewed in the selected publications . However, the protein composition in a human tissue, cell, or body‐fluid proteome is very complex with a very wide abundance range and complicated huge protein speciation caused by splicing, truncation and modification .…”

Preprocessing of 2-Dimensional Gel Electrophoresis Images Applied to Proteomic Analysis: A Review

“…2-DGE was initially employed for protein separation and analysis approximately in 1975 [5] . Even though it was introduced more than four decades ago, nowadays 2-DGE is still widely used for whole proteome analysis [6] , [7] , comparative analysis of proteome changes [6] , [8] , biomarker discovery, cancer research [9] , [10] , as well as for the identification of protein isoforms and post-translational modifications [11] , among other purposes. Its popularity could be attributed to several factors.…”