Mass spectrometric characterization of mitochondrial complex I NDUFA10 variants.

In the present study, we have used a combination of 2-DE and MS to isolate and characterize two variants of the mitochondrial complex I subunit NDUFA10 from Wistar rat brain. Extensive MS/MS analysis revealed that a D/N substitution at position 120 resulting from a 353A/G transition in the coding gene is the biochemical difference between the two most abundant NDUFA10 isoforms. Moreover, 33 modifications of distinct chemical nature targeting 59 specific residues were found to be common to the acidic and basic forms. Positions C67, H149 and H322 of NDUFA10 were specially targeted by different modifications suggesting the high reactivity of these residues and their potential implication in the regulation of the protein function. Together with nonenzymatic modifications that can form in the sample isolation and workup steps, such as oxidation of methionine, tryptophan, cysteine and histidine, we describe amino acid variants of unknown chemical structure that must be further characterized, as well as accumulation of R, K and H methylations and probably K acetylations at the C-terminal region that might play a role in the control of NDUFA10 activity according to similar mechanisms to those described for histones.

Detection of artifacts and peptide modifications in liquid chromatography/mass spectrometry data using two-dimensional signal intensity map data visualization.

We demonstrate how visualization of liquid chromatography/mass spectrometry data as a two-dimensional signal intensity map can be used to assess the overall quality of the data, for the identification of polymer contaminants and artifacts, as well as for the confirmation of post-translational modifications.

Reproducibility of LC-MS-based protein identification.

Traditional analysis of liquid chromatography-mass spectrometry (LC-MS) data, typically performed by reviewing chromatograms and the corresponding mass spectra, is both time-consuming and difficult. Detailed data analysis is therefore often omitted in proteomics applications. When analysing multiple proteomics samples, it is usually only the final list of identified proteins that is reviewed. This may lead to unnecessarily complex or even contradictory results because the content of the list of identified proteins depends heavily on the conditions for triggering the collection of tandem mass spectra. Small changes in the signal intensity of a peptide in different LC-MS experiments can lead to the collection of a tandem mass spectrum in one experiment but not in another. Also, the quality of the tandem mass spectrometry experiments can vary, leading to successful identification in some cases but not in others. Using a novel image analysis approach, it is possible to achieve repeat analysis with a very high reproducibility by matching peptides across different LC-MS experiments using the retention time and parent mass over charge (m/z). It is also easy to confirm the final result visually. This approach has been investigated by using tryptic digests of integral membrane proteins from organelle-enriched fractions from Arabidopsis thaliana and it has been demonstrated that very highly reproducible, consistent, and reliable LC-MS data interpretation can be made.

Photochromic chromopeptides derived from phycoerythrocyanin: biophysical and biochemical characterization.

Truncated chromopeptides have been prepared from the small photo- and redox-switchable biliprotein alpha-phycoerythrocyanin (alpha-PEC). The native chromoprotein consists of a C-terminal globin domain containing the chromophore and the regulatory cysteins 98 and 99, and a two-helix (X,Y) N-terminal domain responsible for aggregation. Digestion with chymotrypsin-free trypsin leads to three chromopeptides, (N-30, N-33 and N-35), basically lacking the two N-terminal helices X and Y. The photo- and redox chemistry of the major product (N-33) is identical, qualitatively and quantitatively, to that of native alpha-PEC. A series of N- and C-terminally truncated polypeptides were expressed in E. coli and subjected to autocatalytic and enzymatic reconstitution with phycocyanobilin. Enzymatic reconstitution was possible with N-terminally truncated polypeptides up to 45 aa, while neither a more extensively shortened (N-63) peptide, nor two C-terminally shortened polypeptides could be reconstituted. All chromopeptides recovered from enzymatic reconstitution contained the native phycoviolobilin chromophore and showed the photochemical and redox reactivity of alpha-PEC, albeit quantitatively reduced in the N-45 chromopeptide.

Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides.

The alpha-phycoerythrocyanin subunits of the different phycoerythrocyanin complexes of the phycobilisomes from the cyanobacterium Mastigocladus laminosus perform a remarkable photochemistry. Similar to phytochromes - the photoreceptors of higher plants - the spectral properties of the molecule reversibly change according to the irradiation wavelength. To enable extensive analyses, the protein has been produced at high yield by improving purification protocols. As a result, several comparative studies on the Z- and E-configurations of the intact alpha-subunit, and also on photoactive peptides originating from nonspecific degradations of the chromoprotein, were possible. The analyses comprise absorbance, fluorescence and CD spectroscopy, crystallization, preliminary X-ray measurements, mass spectrometry, N-terminal amino acid sequencing and 1D NMR spectroscopy. Intact alpha-phycoerythrocyanin aggregates significantly, due to hydrophobic interactions between the two N-terminal helices. Removal of these helices reduces the aggregation but also destabilizes the protein fold. The complete subunit could be crystallized in its E-configuration, but the X-ray measurement conditions must be improved. Nevertheless, NMR spectroscopy on a soluble photoactive peptide presents the first insight into the complex chromophore protein interactions that are dependent on the light induced state. The chromophore environment in the Z-configuration is rigid whereas other regions of the protein are more flexible. In contrast, the E-configuration has a mobile chromophore, especially the pyrrole ring D, while other regions of the protein rigidified compared to the Z-configuration.