Toward the Complete Yeast Mitochondrial Proteome:  Multidimensional Separation Techniques for Mitochondrial Proteomics

Abstract

Proteomic analyses of different subcellular compartments, so-called organellar proteomics, facilitate the understanding of cellular functions on a molecular level. In this work, various orthogonal multidimensional separation techniques both on the protein and on the peptide level are compared with regard to the number of identified proteins as well as the classes of proteins accessible by the respective methodology. The most complete overview was achieved by a combination of such orthogonal techniques as shown by the analysis of the yeast mitochondrial proteome. A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS, 1D-SDS-PAGE combined with nano-LC-MS/MS and 2D-PAGE with subsequent MALDI-mass fingerprinting. Our PROMITO approach identified the 749 proteins, which were found in the largest previous study on the yeast mitochondrial proteome, and additionally 102 proteins including 42 open reading frames with unknown function, providing the basis for a more detailed elucidation of mitochondrial processes. Comparison of the different approaches emphasizes a bias of 2D-PAGE against proteins with very high isoelectric points as well as large and hydrophobic proteins, which can be accessed more appropriately by the other methods. While 2D-PAGE has advantages in the possible separation of protein isoforms and quantitative differential profiling, 1D-SDS-PAGE with nano-LC-MS/MS and multidimensional LC-MS/MS are better suited for efficient protein identification as they are less biased against distinct classes of proteins. Thus, comprehensive proteome analyses can only be realized by a combination of such orthogonal approaches, leading to the largest dataset available for the mitochondrial proteome of yeast.

The Cardiolipin Transacylase, Tafazzin, Associates with Two Distinct Respiratory Components Providing Insight into Barth Syndrome

“…First, the unidentified proteins that participate in these low-molecular-weight Taz1p complexes may not be detected by the used proteomic strategy (SDS-PAGE followed by LC-MS/MS). It is well documented that different proteomic strategies provide distinct information (Reinders et al, 2006); thus complimentary downstream analyses after CNAP may facilitate identification of these partner proteins. Second, the low-molecular-weight Taz1p complexes may represent Taz1p in association with phospholipids and/or their derivatives.…”

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

“…While 2-D PAGE has been employed toward an inventory of the mitochondrial proteome [7,11,[15][16][17][18][19], mitochondria contain many membrane-bound and very basic proteins that are quite difficult to be analyzed by 2-D PAGE [7,14]. 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].…”

“…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.…”

Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism

“…Recent proteomic studies suggest that there are as many as 1,000 mitochondrial proteins in Saccharomyces cerevisiae (1,2) and 1,500 in mammals (3,4). The majority of these mitochondrial proteins are encoded by the nuclear genome and translated by cytosolic ribosomes, except for a few that are encoded by the mitochondrial genomic DNA (5,6).…”

“…Therefore, the import receptor Tom70 serves as a docking site for cytosolic multichaperone complexes that contain preprotein and the co-chaperones DNAJA1 and DNAJA2 (10,12). This chaperone-docking step is considered essential for the initiation of the Tom70-mediated import pathway where the preprotein is in direct contact with Tom70 (10,13 2 The abbreviations used are: TOM, translocase of the outer mitochondrial membrane; ANT, adenine nucleotide transporter; GIP, general import pore; ISP, Rieske iron-sulfur protein; OTC, ornithine transcarbamylase; PiC, phosphate carrier; SPR, surface plasmon resonance; s-SMCC, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate; TPR, tetratricopeptide repeat; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid.…”