MS-specific noise model reveals the potential of iTRAQ in quantitative proteomics.

MOTIVATION: Mass spectrometry (MS) data are impaired by noise similar to many other analytical methods. Therefore, proteomics requires statistical approaches to determine the reliability of regulatory information if protein quantification is based on ion intensities observed in MS. RESULTS: We suggest a procedure to model instrument and workflow-specific noise behaviour of iTRAQ reporter ions that can provide regulatory information during automated peptide sequencing by LC-MS/MS. The established mathematical model representatively predicts possible variations of iTRAQ reporter ions in an MS data-dependent manner. The model can be utilized to calculate the robustness of regulatory information systematically at the peptide level in so-called bottom-up proteome approaches. It allows to determine the best fitting regulation factor and in addition to calculate the probability of alternative regulations. The result can be visualized as likelihood curves summarizing both the quantity and quality of regulatory information. Likelihood curves basically can be calculated from all peptides belonging to different regions of proteins if they are detected in LC-MS/MS experiments. Therefore, this approach renders excellent opportunities to detect and statistically validate dynamic post-translational modifications usually affecting only particular regions of the whole protein. The detection of known phosphorylation events at protein kinases served as a first proof of concept in this study and underscores the potential for noise models in quantitative proteomics.

[Differential therapy in radial head fractures]. / Differenziertes Behandlungskonzept der Radiusköpfchenfraktur.

The therapeutic regimen of radial head fractures, especially of displaced and comminuted types is controversial. The radial head resection has been critically reviewed over the past years. From 1984-1993 and 1996-1999, 105 radial head fractures were treated in our hospital. 74 were subject to clinical and radiological follow-up. Fracture-types were classified according to Mason. Undisplaced fractures were treated conservatively, displaced 2-fragment-fractures by an open reduction and screw fixation, and multifragment-fractures by a radial head resection. The results were studied on a functional and radiological basis using the "Functional Rating Index" of Broberg and Morrey and the radiological Score of Albrecht and Ganz. After conservative therapy over 80 % achieved excellent and good as well as 12.5 % satisfactory and 6.3 % unsatisfactory results. After reduction and internal fixation again 80 % had excellent and good results. After radial head resection excellent and good results were achieved in 54.6 % of the cases, satisfactory results in 24.2 % and in 21.2 % unsatisfactory results, however prognosis-influencing concomitant injuries were often present in the latter group. Using the right indication and technique, the radial head resection still is a recommendable therapeutic procedure with an altogether good prognosis. This especially applies to isolated radial head fractures where excellent and good results can be achieved in approximately 70 %.

Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.

Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20.

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.

Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels.

Purification of mitochondria and mitochondrial protein complexes from green tissues is often severely impaired by the presence of chloroplasts and their proteins. Here we present a method which allows analysis of respiratory protein complexes from potato leaves. The procedure includes the preparation of an organellar fraction specifically enriched in mitochondria and the separation of organellar protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast protein complexes have been resolved simultaneously in a native gel. BN-PAGE allowed the separation of eleven bands, including the mitochondrial NADH-dehydrogenase, the bc1 complex and the mitochondrial F1-ATP synthase as well as the chloroplast F1-ATP synthase, the cytochrome b6f complex, the two photosystems and the light harvesting complex. The resolution of the protein complexes in the first dimension was good enough to allow identification of all subunits of individual complexes in the second dimension under denaturing conditions. Thus, BN-PAGE offers an opportunity to analyze mitochondrial and chloroplast protein complexes from a single preparation from very small amounts of tissue. The implications of our findings, for studies on protein expression and turnover in different tissues and developmental stages, are discussed.

Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants.

Transport of most nuclear encoded mitochondrial proteins into mitochondria is mediated by heteropolymeric translocases in the membranes of the organelles. The translocase of the outer mitochondrial membrane (TOM) was characterized in fungi, and it was shown that TOM from yeast comprises nine different subunits. This publication is the first report on the preparation of the TOM complex from plant mitochondria. The protein complex from potato was purified by (a) blue native polyacrylamide gel electrophoresis and (b) by immunoaffinity chromatography. On blue native gels, the potato TOM complex runs close to cytochrome c oxidase at 230 kDa and hence only comprises about half of the size of fungal TOM complexes. Analysis of the TOM complex from potato by SDS-polyacrylamide gel electrophoresis allows separation of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa. The 23-kDa protein is identical to the previously characterized potato TOM20 receptor, as shown by in vitro assembly of this protein into the 230-kDa complex, by immunoblotting and by direct protein sequencing. Partial amino acid sequence data of the other subunits allowed us to identify sequence similarity between the 36-kDa protein and fungal TOM40. Sequence analysis of cDNAs encoding the 7-kDa protein revealed significant sequence homology of this protein to TOM7 from yeast. However, potato TOM7 has a N-terminal extension, which is very rich in basic amino acids. Counterparts to the TOM22 and TOM37 proteins from yeast seem to be absent in the potato TOM complex, whereas an additional low molecular mass subunit occurs. Functional implications of these findings are discussed.

New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria.

Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'blue native-polyacrylamide gel electrophoresis' (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN-PAGE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.

Cytochrome c reductase from potato does not comprise three core proteins but contains an additional low-molecular-mass subunit.

Analysis of cytochrome c reductase from potato by Tricine/SDS/PAGE reveals 10 bands representing 10 different subunits. In comparison to glycine/SDS/PAGE one additional small protein becomes visible, whereas the three large core proteins are not resolved. The identity of the subunits was determined by immunoblotting and direct sequence determination. Sequence data for the novel small component were used to derive oligonucleotides for probing a potato cDNA-library and isolating corresponding clones. The newly identified subunit is a 6.7-kDa protein, that exhibits significant sequence similarity to a 8.5-kDa subunit of cytochrome c reductase from yeast and the 6.5-kDa iron-sulfur-protein-binding factor from the equivalent enzyme complex from beef. Also the potato 6.7-kDa subunit can be dissociated from the cytochrome c reductase complex together with the iron-sulfur protein. To address the question of whether three or two core subunits occur simultaneously in monomeric cytochrome c reductase complexes from potato, a peptide-specific antibody was generated. The antiserum is capable of discriminating between the 55-kDa and 53-kDa core proteins, which can be separated by glycine/SDS/PAGE and which were previously found to be structurally related. Immunoprecipitations of isolated cytochrome c reductase from potato using this antibody revealed an enzyme complex containing only two core proteins. The simultaneous occurrence of only two core subunits was confirmed by a comparison of the molecular masses of cytochrome c reductase from potato and beef by blue-native-gel electrophoresis. Hence the cytochrome c reductase complexes from potato, beef and yeast have a very conserved subunit composition. The evolutionary implications of these findings are discussed.

The 'Hinge' protein of cytochrome c reductase from potato lacks the acidic domain and has no cleavable presequence.

The 'Hinge' protein of cytochrome c reductase from fungi and mammals is thought to support electron transport from cytochrome c1 to cytochrome c and was reported to be one of the most acidic proteins known. Isolation and analysis of cDNA clones of the first 'Hinge' protein from a plant source reveals that it has a surplus of basic residues in potato. While the overall identity between the deduced amino acid sequence of the potato 'Hinge' protein and the proteins from yeast and bovine is in the range of 40%, the characteristic acidic domain is lacking. Therefore the numerous theories on the function of the mitochondrial 'Hinge' protein seem not to apply for the protein from potato. Also the atypical acidic presequence of the 'Hinge' protein from fungi and mammals is absent as revealed by N-terminal sequencing of the isolated potato 'Hinge' protein. Functional implications of these results for the 'Hinge' proteins from other organisms are discussed.