An Alternative Strategy To Determine the Mitochondrial Proteome Using Sucrose Gradient Fractionation and 1D PAGE on Highly Purified Human Heart Mitochondria

Abstract

An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family.

Mitochondria: A mirror into cellular dysfunction in heart disease

“…Once released from the cardiomyocyte, mitochondria can be partitioned using discontinuous gradients including sucrose, percoll/metrizamide, which exploit the density of mitochondria to facilitate further enrichment. Such enrichment strategies have been used for proteomic investigations [4,5,[73][74][75][76][77][78][79][80][81][82], with varying degrees of myofibrillar contamination. The study conducted by Forner et al [4] also investigated within the mitochondrion, observing IMM and matrix proteins across multiple tissue types including myocardium.…”

“…This approach has been utilized by gel-free proteomic studies to increase the coverage of the mitochondrial proteome [4,84]. Alternatively, integral protein-protein interactions [81], and protein affinity [86] have been utilized to improve visualization of the dynamic range of mitochondrial proteome. By increasing the depth of coverage, the goal of obtaining a truly global understanding of the mechanisms and pathways altered in the mitochondria with CV disease becomes more likely.…”

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

“…Digests were prepared for MALDI-TOF peptide mass fingerprinting, and spectra were automatically acquired on a Voyager DE-STR TM as reported previously (36). Peptide mass fingerprints from baseline-corrected, noise-filtered, and de-isotoped peaks were then analyzed using the program Protein Prospector, MS-Fit (37) with and without the Intellical algorithm from Applied Biosystems as described previously (37,38).…”

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

“…Consequently, considerable efforts have been devoted to the application of various LC techniques in single or multidimensional separation format prior to MS detection for the analysis of mitochondrial proteins [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. In particular, the peptide-based shotgun proteomic studies fully exploit the resolution and sensitivity achievable with multidimensional LC-MS or SDS-PAGE/LC-MS approaches, allowing many additional mitochondrial proteins to be identified.…”