Top-Down Proteomics and Comparative 2D-DIGE Analysis

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

“…The development of the differential imaging gel electrophoresis technique for increased sensitivity and reproducibility using fluorescent dyes proved to be a significant addition to sample analysis using 2D-GE [ 86 , 87 , 88 ]. Following optimized sample preparation [ 89 ], fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is based on labeling of protein within a sample with a different fluorophore (CyDye3, CyDye5 or CyDye2) that binds covalently with the epsilon amino group of lysine residues for minimal labeling [ 90 ]. Typically, the internal control is a combination of all samples that will be analyzed within a single experiment and is labeled with CyDye2.…”

“…Software packages including DeCyder, SameSpots and Dymension 3 can be incorporated into the workflow to aid in the identification of significant proteins [ 94 , 95 ]. Saturation labeling using CyDye3 and CyDye5 fluorophores with maleimide chemistry can be employed to label all cysteine residues within the sample of interest [ 96 ], enhancing sensitivity compared to the minimal approach [ 90 ]. As an alternative to gel-based protein separation, multi-dimensional protein identification technology (MudPIT) [ 97 ] can be employed, which is based on two-dimensional liquid chromatography (2D-LC) followed by MS-based analysis [ 98 ].…”

“…Table 1 lists major proteomic techniques, as well as a variety of biochemical, immunochemical and cell biological methods that can be used to independently verify the MS-based identification of specific proteoforms or the findings of changes in protein abundance from comparative proteomic studies. These methods are frequently employed to study cell or tissue specimens derived from normal versus diseased skeletal muscles, including the following: Two-dimensional gel electrophoresis (2D-GE) [ 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 71 ]; Differential imaging gel electrophoresis (2D-DIGE) [ 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 ]; Specialized gel-based methods for studying specific protein species [ 67 , 68 , 69 , 77 , 78 , 79 , 80 ]; Gel electrophoresis–liquid chromatography methods (GeLC) [ 103 , 104 , 105 ]; Protein microarrays [ 143 , 144 , 145 , 146 ]; Sample preparation for proteomic analysis [ 89 , 113 , 114 , …”

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…High-throughput comparative studies can be performed via comparative two-dimensional fluorescence gel electrophoresis (CoFGE) [124,125]. Difference gel electrophoresis (2D-DIGE) can be carried out with 2-CyDye or 3-CyDye systems using saturation versus minimal labelling approaches [126,127]. Of note, 2D-GE, including the 2D-DIGE technique, can be employed effectively to study native protein configurations [128,129], as well as PTMs using specific chemical stains or blot overlay methods.…”

“…x FOR PEER REVIEW 4 of 36 minimal labelling approaches [126,127]. Of note, 2D-GE, including the 2D-DIGE technique, can be employed effectively to study native protein configurations [128,129], as well as PTMs using specific chemical stains or blot overlay methods.…”