In this study, we combined SILAC labeling, MudPIT separation of protein/peptide, including SDS-PAGE, and tandem MS to characterize the mitochondrial proteome of MES cells as well as the alterations in the mitochondrial proteome in the presence of rotenone. Several major findings have been found from this investigation, including: 1) a total of 1722 proteins were identified as mitochondrial protein or mitochondria-associated proteins, which is the most comprehensive study of the mitochondrial proteome so far; 2) quantitative analysis of the mitochondrial protein profiles identified 311 proteins that displayed changes in relative abundance in MES cells treated with rotenone vs. controls. Among these, 110 proteins have been represented in the MitoP2 database as mitochondrial proteins; and 3) a subset of novel mitochondria-associated proteins affected by rotenone was validated by confocal microscopy and WB to assess their localization in the mitochondria as well as quantitative changes as determined by quantitative proteomics.
The first achievement of this study is the comprehensive survey of mitochondrial proteins in MES cells, a DAergic cell line widely used for the investigation of PD pathogenesis [26
]. Not only does extensive characterization of the mitochondrial proteome expand current knowledge regarding the profiles of mitochondrial proteins, it also supplies the necessary information to appropriately interpret the mechanisms of rotenone-induced mitochondrial dysfunction in PD. Comparing the results reported in the literature [25
], the current analysis is a much deeper analysis into the mitochondrial proteome in a neuronal cell line, largely owing to better sample separation by SDS-PAGE and LC, i.e. extensive fractionation prior to MS analysis. This study, using only 80–100 μg of mitochondrial proteins as the starting material, followed by simple LC-MS/MS analysis of 10 gel slices in two independent experiments, afforded the conclusive identification of 1864 proteins. Thus, this simple technology seems to be the most effective method of identifying a large number of proteins from different cell types. Furthermore, this is the first large-scale study where the mitochondrial proteome from the MES cell line was carefully characterized, providing organelle-specific proteomics information for these cells relevant to PD pathogenesis. Among those proteins identified, approximately 50% did not have an apparent orthologue in the MitoP2 database. Several reasons are likely responsible for this absence in the database. First, the current database is still far from complete and most of the missing proteins are hypothetical proteins or proteins with unknown functions. Second, although the purification protocols for mitochondria have been refined by several groups over many years, the exquisite sensitivity of modern mass spectrometers has revealed that it is very difficult, if not impossible, to purify these or any other organelles to homogeneity. In our study, a small portion of those proteins (7.5%) was considered as contaminated from cytoplasm, nucleus, ER, golgi or lysosome, based on the functional interpretation of SWISS-Prot database. While the sucrose gradient ultracentrifugation technique we used to isolate the mitochondrial fraction is widely recognized as the technique producing least contamination [30
], it remains likely that at least some of these proteins, particularly ER proteins, were co-localized in the mitochondrial fraction as contaminants. It is also possible that some of the "contaminating" proteins are partially localized in mitochondria or interact with other mitochondrial proteins. More detailed studies such as immuno-colocalization experiments would be required to distinguish these different possibilities.
Next, to better understand the mechanisms underlying mitochondrial dysfunction induced by rotenone, we used SILAC to compare the protein profiling of mitochondria in rotenone-treated MES cells versus controls. With SILAC methods, a total of 156 and 155 proteins showed significant increase or decrease after rotenone treatment. Among these, 53 and 57 proteins have been present in the MitoP2 database as mitochondrial proteins respectively. One of the major caveats of proteomics identification of proteins relates to the fact that proteins are inferred by the peptide sequence based on databases, which are currently incomplete. Thus, any given identification, with or without quantitative changes, should be considered provisional until it can be validated via alternative means. It is obviously impractical to validate the entire list of proteins listed in Additional file 1
or even Table ; to this end, we validated five candidate proteins (sacsin, SNX1, VAMP3, MAP2K2 and SHH) in terms of their cellular location as well as quantitative alternation after rotenone treatment. Two of these proteins, SHH and SNX1, were co-localized to mitochondria convincingly by confocal investigation. Three of these proteins, SNX1, sacsin and VAMP3, when assessed by WB analysis, showed good agreement with our SILAC results. It should be noted that there are many caveats associated with WB and immnocytochemistry, such that a negative result by these assays does not necessarily negate what was observed by quantitative proteomics.
The biological importance of the three validated proteins in PD or rotenone pathogenesis is largely unknown. A potential role of sacsin has been implicated in an early onset neurodegenerative disorder, autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which occurs with a high prevalence in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec [32
]. Sacsin contains three regions with Hsp90 subdomains that have similarity to each other and the protein product of an Arabidopsis thaliana
ORF. The presence of heat shock domains in the sacsin protein suggests its possible function in chaperone-mediated protein folding [32
]. SNX1 is a protein that binds to the cytoplasmic domain of plasma membrane receptors [34
]. It has been proposed to be associated with early endosomes and the regulation of endocytic trafficking of plasma membrane proteins in early endosomes such as epidermal growth factor receptor (EGFR) [35
] and protease-activated receptor-1 (PAR1) [36
]. VAMP3, an early endosomal vesicular soluble N-ethylmaleimide-sensitive protein attachment protein receptor (v-SNARE), is a membrane trafficking protein of a constitutively recycling pathway [37
]. Although neither SNX1 nor VAMP3 has been linked to PD pathogenesis thus far, it has been clearly demonstrated by others that dysfunctions of intracellular trafficking and lysosomal degradation are involved in PD pathogenesis [38
]. Finally, it needs to be pointed out that this investigation is limited in that we have only compared the protein profiles in MES cells treated with rotenone for three days, when LB like inclusions are found [17
]. It would be important in future experiments to examine earlier time points, revealing factors/pathways leading to cell death prior to formation of LB like inclusions.