An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis

Abstract

Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.

Subcellular proteomics of mice gastrocnemius and soleus muscles

“…Comparing our data with those published previously by Gelfi and coworkers [9] and Jarrold and coworkers [14], a considerably greater number of identified proteins were achieved, amounting to 108 identified proteins in the case of the gastrocnemius muscle and to 103 in the case of the soleus muscle. Accordingly, gel maps of the nuclear fraction from gastrocnemius and soleus muscles present several spots identified as cytoskeletal components, for example, myosins, tubulin, and actin-binding proteins that constitute approximately 47% of the total number of identified proteins.…”

“…In consequence, proteins such as c-myc, eukaryotic translation elongation factor 1, and eukaryotic translation initiation factor 5A were identified for the first time in the nuclear fraction of both gastrocnemius and soleus muscles using proteomics. The additional centrifugation of the cytosolic fraction induces the reduction of cytoskeletal and mitochondrial contributions.Comparing our data with those published previously by Gelfi and coworkers [9] and Jarrold and coworkers [14], a considerably greater number of identified proteins were achieved, amounting to 108 identified proteins in the case of the gastrocnemius muscle and to 103 in the case of the soleus muscle. Accordingly, gel maps of the nuclear fraction from gastrocnemius and soleus muscles present several spots identified as cytoskeletal components, for example, myosins, tubulin, and actin-binding proteins that constitute approximately 47% of the total number of identified proteins.…”

“…In this regard, the soleus and the white portion of gastrocnemius of mice are composed mainly of fast-and slow-twitch muscle fibers, respectively [5][6][7], and these two muscles have been used as typical models in proteomics. During the past few years, several studies have been performed using two-dimensional gel electrophoresis (2-DE) 1 combined with mass spectrometry (MS) to characterize skeletal muscle protein composition of typical slow-and fast-twitch skeletal muscles [8][9][10][11][12][13][14][15][16][17]. In a recent proteomics study on murine gastrocnemius and soleus muscle extracts, performed by Gelfi and coworkers [9], more than 800 spots on each 2-DE were detected by silver staining, leading to the identification of 85 different proteins belonging to the most abundant structural and metabolic protein classes.…”

“…Despite the large number of visualized spots by 2-DE, proteins with lower relative abundances, usually involved in protein biosynthesis and cell stress response, are probably masked by structural or metabolic proteins [18,19]. To counteract this, Jarrold and coworkers [14] performed the depletion of abundant muscle proteins by a high pH treatment followed by 2-DE analysis, resulting in the elimination of the major contractile proteins and in the detection of nine minor proteins, mainly belonging to mitochondria, for the first time. Nevertheless, the most often used strategy to reduce sample complexity is based on subcellular fractionation [19][20][21], which to our knowledge has never been done for muscle proteomics characterization.…”

Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice

“…Differential expression between the groups was determined as a fold change, and proteins with the most appropriated change in expression between the groups were selected for analysis by peptide mass spectrometry (MS). Protein spots were excised, chopped, destained, and dehydrated from the gels and subjected to a tryptic digestion, as originally described by Shevchenko et al (40) with modification as presented by Jarrold et al (15). The extracted peptides were concentrated in a speed vac centrifuge (Speed-Vac plus, Savant, Ramsey, MN) to a final volume of 10 -15 l. Peptides were desalted and purified utilizing the C18 ZipTips (Millipore, Bedford, MA).…”