Brainstem deficiency of the 14-3-3 regulator of serotonin synthesis: a proteomics analysis in the sudden infant death syndrome.

Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.

Proteorhodopsin in the ubiquitous marine bacterium SAR11.

Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 ('Pelagibacter ubique'), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.

Proteomic analysis of novel marine bacteria using MALDI and ESI mass spectrometry.

The objective of this study was to develop a mass spectrometric protocol to search for proteins related to phototrophy in marine bacteria. The genes that produce proteins involved in conversion of light into energy have been detected by cloning-sequencing from some of these bacteria, but it was previously unknown if these proteins were actually expressed. Attaining this study's goal was complicated by the fact that the samples consisted of miniscule cell pellets, which yielded small amounts of very complex mixtures of proteins. Sample preparation and analysis were tailored to optimize the probability of detecting the proteins of interest. It has been reported that using both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) to analyze a mixture of peptides leads to the identification of more peptides that either technique alone. In order to exploit this complementarity between ESI and MALDI for proteomic analysis, samples were analyzed using both ionization techniques. With correct choices in sample preparation and ionization process, biologically relevant proteins can be identified out of small samples containing whole proteomes.

Polyphyletic photosynthetic reaction centre genes in oligotrophic marine Gammaproteobacteria.

Ecological studies indicate that aerobic anoxygenic phototrophic bacteria (AAP) that use bacteriochlorophyll to support phototrophic electron transport are widely distributed in the oceans. All cultivated marine AAP are alpha-3 and alpha-4 Proteobacteria, but metagenomic evidence indicates that uncultured AAP Gammaproteobacteria are important members of ocean surface microbial communities. Here we report the description of obligately oligotrophic, marine Gammaproteobacteria that have genes for aerobic anoxygenic photosynthesis. Three strains belonging to the OM60 clade were isolated in autoclaved seawater media. Polymerase chain reaction assays for the pufM gene show that these strains contain photosynthetic reaction centre genes. DNA sequencing and phylogenetic analysis indicate that the pufM genes are polyphyletic, suggesting multiple instances of lateral gene transfer. Peptide sequences from six photosynthesis genes (pufL, pufM, pufC, pufB, pufA and puhA) were detected by proteomic analyses of strain HTCC2080 cells grown aerobically in seawater. They closely match predicted peptides from an environmental seawater bacterial artificial chromosome clone of gammaproteobacterial origin, thus identifying the OM60 clade as a significant source of gammaproteobacterial AAP genes in marine systems. The cell yield and rate of growth of HTCC2080 in autoclaved, aerobic seawater increased in the light. These findings identify the OM60 clade as a source of Gammaproteobacteria AAP genes in coastal oceans, and demonstrate that aerobic, anoxygenic photosynthetic metabolism can enhance the productivity of marine oligotrophic bacteria that also grow heterotrophically in darkness.

Complementary use of MALDI and ESI for the HPLC-MS/MS analysis of DNA-binding proteins.

Proteins from Escherichia coli were isolated based on their ability to bind DNA and digested in-solution with trypsin; the resulting peptides were separated using HPLC and subsequently analyzed using MALDI TOF/TOF and ESI Q-TOF instruments. Various properties of the peptides observed with the two ionization techniques were compared taking into account the differences between the mass analyzers. This empirical analysis of a data set containing hundreds of peptides and thousands of individual amino acids supports some of the currently held notions regarding the complementary nature of the two ionization processes. Specifically, ESI tends to favor the identification of hydrophobic peptides whereas MALDI tends to lead to the identification of basic and aromatic species. Findings from the present study suggest that ESI and MALDI may be complementary due to the biases of the two ionization techniques for certain classes of amino acids. From a practical standpoint, these biases indicate that, for the present at least, analyses must be performed on both types of instruments in order to gain the most information possible out of a given set of samples in a proteomics study.

Alkylation of protein disulfide isomerase by the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane and mass spectrometric characterization of the adducts.

The reactivity of the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG), the glutathione conjugate of 1,2-dichloroethane, with the catalytic sites of protein disulfide isomerase (PDI) was investigated. The two cysteine residues of the two active sites of PDI are expected to be the major targets of alkylation. PDI was incubated with equimolar to 100-fold excess CEG. The activity of PDI was irreversibly inhibited with a concurrent loss of two thiols; however, PDI oxidative refolding activity was not completely inhibited. With mass spectrometry, sequencing PDI identified one alkylation event on each of the N-terminal cysteine residues in the two active site peptides. PDI appears robust and able to maintain some activity by steric constraint. We have established that the episulfonium ion of CEG can adduct PDI and may have important toxicologic significance for 1,2-dichloroethane toxicity.

Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry.

Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser(55), Ser(66), and Ser(472)) and NFM has 22 (Ser(512), Ser(546), Ser(554), Ser(560), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), Ser(750), Ser(756), Ser(770), and Ser(846)) and two tentative sites (Ser(659)/Thr(661) and Thr(840)). Ser(66) was previously not known to be phosphorylated in NFL of other species, while two sites (Ser(55) and Ser(472)) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser(55), Ser(66), Ser(472), are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser(512), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), and Ser(750)), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser(546), Ser(554), Ser(560), Ser(756), Ser(770), and Ser(846)). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents.

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.