Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 78
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The addition of increasing amounts of cytosol tendedto suppress the phosphorylation of p46, implying the presence of an inhibitory cytosolic factor that was diminished after ischemia (Fig. 16.3).These studies indicated that the 46 kDa protein was a genuine mitochondrialphosphoprotein regulated by cytosolic signals. Specifically, the 46 kDa protein thatwe had demonstrated to be phosphorylated in the reconstituted system met thefollowing criteria:1. Differentially phosphorylated (greater in ischemia than control)2. Resident in mitochondria (including mitochondria with intact membranes)3.
Present in cardiomyocytes.These findings provided the impetus to pursue identification of the 46 kDa mitochondrial protein as a target of phosphorylation in the setting of ischemia/reperfusion.Our initial approach to purify the 46 kDa protein was to use FPLC. The mitochondria were lysed in detergent-containing buffer and the soluble fraction wasresolved on an anion exchange column. The 46 kDa protein eluted in multiplefractions, with excessive sample dilution. As we discovered later, this was probablydue in part to the fact that p46 was multiply phosphorylated.
We were unsuccessful in eluting the phosphoprotein from a hydrophobic interaction column. Weturned instead to 2D gel electrophoresis. While many of the mitochondrial proteins are rather hydrophobic and require special treatment in order to successfullysolubilize them, we were fortunate in that the 46 kDa phosphoprotein was readilysolubilized and resolved nicely on a conventional IEF strip and second dimensionSDS-PAGE.2D spot mapping can be quite difficult, depending upon the complexity of thesample under analysis.
We were optimistic because the autoradiogram of mitochondria after in vitro labeling was deceptively simple, revealing only *20 spots.Encouragingly, the 46 kDa band resolved into *3 closely spaced spots differingby isoelectric point (pI *6.5), and which were nicely separated from other phosphoproteins. However, the complexity was greatly increased when we prepared aCoomassie-stained gel of the same material. Worse yet, the 46 kDa spots were notabundant enough to be detected by Coomassie staining of a gel loaded with up to2 mg of mitochondrial protein. At this point, we were using total mitochondria.3Fig. 16.4 Preparation of sample for identification by mass spectrometry. Inner mitochondrial membrane from 7 mg of mitochondriawere used for phosphorylation with “cold” or‘hot’ ATP and resolved by 2DE for Coomassiestaining and autoradiography, respectively.
Inset shows the three spots of interest, whichwere removed and separately subjected totrypsin digestion and MALDI-MS. The middlespot was subsequently used for MS/MS analysis, shown on the right. Reproduced with permission from Circulation Research 89:461–467,2001.28328416 Phosphoproteins in Heart MitochondriaHowever, we reasoned that if we fractionated the mitochondria further, we couldreduce the complexity and could also enrich for the 46 kDa protein.
Submitochondrial fractionation revealed that the 46 kDa protein was present in the matrix andin the inner membrane.Because it was easier to prepare and concentrate the inner membrane (IM), weused this as the material for subsequent analysis. This sub-fractionation was sufficient to enrich the 46 kDa protein so that it could be detected by Coomassie staining after loading IM derived from 7 mg of mitochondria on a large (18 cm) preparative gel. While having enough protein to detect by Coomassie staining is notessential, it is a reassuring indication that one will have enough material for successful identification by mass spectroscopy.
Moreover, it will reveal the presence ofadditional contaminating proteins in the neighborhood that may complicate theanalysis. In order to prepare the 46 kDa phosphoprotein for mass spectrometry,we prepared mitochondria, split into two aliquots, and performed two identicalphosphorylation reactions, one with c32P-ATP and the other with non-radioactiveATP. The mitochondria were then washed and fractionated to obtain inner membranes. We then ran two identical 2-D gels and overlaid the autoradiogram of oneon the Coomassie-stained non-radioactive gel, and cut out the three closely spacedspots of interest. We were fortunate that the region of interest did not have a lotof additional proteins (Fig.
16.4).Karoline Scheffler and Brad Gibson at UCSF analyzed the three samples. Analysis of the tryptic peptides by MALDI-MS revealed that the three spots had identical mass signatures, confirming that they represented multiply phosphorylatedforms of the same protein. A total of 12 mass fingerprints were used to query thedatabase, but failed to match anything in the database. This disappointing outcome exemplifies one of the limitations of current analytical software. While thealgorithm can tolerate one amino acid substitution in a fragment, it fails if thereare two or more amino acid substitutions in a single tryptic fragment.
It isfurther confounded if a tryptic cleavage site is missing. Since very little of the rabbit genome is in the database, one must rely on comparisons with other speciessuch as mouse and human. Unfortunately, rabbit diverges just enough to confound such comparisons.It was therefore necessary to sequence some of the more abundant peptide fragments in order to identify the 46 kDa phosphoprotein, using nanoelectrosprayMS/MS (Fig. 16.4).
By this approach, Drs. Scheffler and Gibson were able to obtain sequences from four peptides, consisting of 13, 12, 6, and 4 amino acids. Thetwo longer peptides were subjected to a BLAST search and matched (100%) withthe human and bovine mitochondrial precursor of the elongation factor Tu (EFTumt) (SwissProt accession numbers P49411 and P49410, respectively). We developed primers and used RT-PCR to obtain partial cDNA sequence for rabbit EFTumt. The overall sequence of mitochondrial EF-Tu is highly conserved, demonstrating 92% homology with human and 95% homology with bovine amino acidsequence. Despite this high degree of homology, detection by the mass fingerprintwas unsuccessful. This points to the need to select starting material from a species that is well represented in the database, and also illustrates the value of se-16.1 Experimental Approach and Pitfallsquencing the genomes of additional species that serve as important animal models, including rabbit, dog, and pig.Having identified the three Coomassie-stained spots as EF-Tumt, it was necessary to verify that the phosphoprotein was indeed EF-Tumt, since it was possiblethat we had instead identified a more abundant protein that colocalized with the46 kDa phosphoprotein.
We were fortunate that antibody to EF-Tumt had beengenerated by Dr. Linda Spremulli, at the University of North Carolina, ChapelHill [4]. We collaborated with Dr. Spremulli in the next phase of the work. Themost straightforward approach would have been to use anti-EF-Tu antibody to immunoprecipitate the phosphoprotein. Unfortunately, EF-Tu is a rather sticky protein and adhered to protein G-Sepharose even in the absence of primary antibody.We therefore resorted to ion exchange chromatography, and found that EF-Tumtand the phosphoprotein eluted in the same fractions. Immunodetection of EF-Tuin those fractions could be superimposed on the autoradiogram of the same blot(Fig.
16.5A). Additionally, the submitochondrial distribution of the 46 kDa phos-Fig. 16.5 Verification that p46 is EF-Tumt.Panel A: Column fractions from anion exchange were concentrated, resolved by SDSPAGE, and subjected to autoradiography(upper panel) and immunoblotting for EF-Tu(lower panel). Panel B: Submitochondrial fractionation was performed to obtain outermembrane (OM), intermembrane space(IMS), inner membrane (IM), and matrix(MTX). Shown are the autoradiogram of p46(32P), immunoblot of mitochondrial EF-Tu/Ts(Tu-Ts), and markers for IM (Rieske iron-sulfur protein, FeS) and matrix (hsp60).
Panel C:2D gel showing autoradiogram of p46 and immunoblot for EF-Tu. Inset shows an enlargement of the immunoblot, showing 2–4 isoelectric variants of EF-Tu from mitochondriaincubated in vitro with cytosol (in vitro) andfrom mitochondria rapidly isolated from afresh heart (in vivo). Reproduced with permission from Circulation Research 89:461-467,2001.28528616 Phosphoproteins in Heart Mitochondriaphoprotein in inner membrane and matrix was identical with the distribution ofEF-Tumt (Fig.
16.5B). Additional evidence came from an immunoblot of a 2-D gel,in which EF-Tu immunoreactivity of three closely spaced spots could be superimposed on the autoradiogram of the phosphoprotein, thus confirming that EF-Tumtexists as multiple isoforms which differ by isoelectric point, presumably representing multiply phosphorylated species (Fig. 16.5C). Comparison of the isoforms ofEF-Tumt observed in the reconstituted preparations of cytosol and mitochondriawith those observed in a freshly isolated heart is shown in the inset (Fig. 16.5C,inset).
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