Expanding the Subproteome of the Inner Mitochondria Using Protein Separation Technologies

Abstract

Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (twodimensional liquid chromatography using the ProteomeLab TM PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one-and two-dimensional LC allowed for the separation of the ␣-subunit of F 1 F 0 ATP synthase that differed due to a change in pI or hydrophobicity. The eukaryotic proteome is a compilation of proteins that represents the integration of numerous cellular processes that begin with the variable transcription of genes to mRNA. These products are then translated to proteins, which may in turn be potentially co-and/or post-translationally modified to produce an array of proteins (1, 2). Due to the large number of unique protein species produced coupled with differences in their relative abundance, there is as of yet no single proteomics technology that has the analytical capacity or sensitivity to realize the goal of complete proteome coverage. One strategy to maximize proteome coverage is to combine synergistic proteomics technologies, particularly if each technology reveals a unique subset of proteins. Using the inner mitochondrial membrane as a model subproteome, we compared the ability of three protein separation methods (two-dimensional LC (2-DLC 1 with the PF2D), one-dimensional reversed phase HPLC (1-DLC; reversed phase high performance liquid chromatography (RP-HPLC)), and two-dimensional gel electrophoresis (2-DE) to determine the relative overlap in protein separation for these technologies.2-DE, a classical proteomics technology that separates proteins based on their pI and molecular weight, has a practical dynamic range of 10 4 orders of magnitude (for r...

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.…”

Delta Protein Kinase C Interacts with the d Subunit of the F1F0 ATPase in Neonatal Cardiac Myocytes Exposed to Hypoxia or Phorbol Ester

“…Briefly, the NCP strips were overlaid with a mixture of purified PKC isozymes and then the overlay strips were probed for and CL) and as is shown, ␦PKC bound only to a protein that co-migrated with dF 1 F 0 immunoreactivity (Fig. 7, panels C versus D) and with a pI (6.5) that has been previously reported for dF 1 F 0 (30). Despite their presence in our two-dimensional blots (Fig.…”

High reproducibility of two-dimensional liquid chromatography using pH-driven fractionation with a pressure-resistant electrode

“…It often restricts the analysis to the most abundant proteins, has demonstrated a relative lack of reproducibility and has trouble resolving proteins of extreme hydrophobicity, mass or isoelectric point (pI) (3,4). To circumvent these limitations and to provide an alternative to 2-DE, liquid-based identified by all technologies (7,8). However, it seems clear that 2D-LC allows the identification of more proteins than 2D-gels and may be more suited for novel protein discovery (7,8).…”

“…To circumvent these limitations and to provide an alternative to 2-DE, liquid-based identified by all technologies (7,8). However, it seems clear that 2D-LC allows the identification of more proteins than 2D-gels and may be more suited for novel protein discovery (7,8).…”