Characterization of Cerebrospinal Fluid via Data-Independent Acquisition Mass Spectrometry.

Cerebrospinal fluid (CSF) is in direct contact with the brain and serves as a valuable specimen to examine diseases of the central nervous system through analyzing its components. These include the analysis of metabolites, cells as well as proteins. For identifying new suitable diagnostic protein biomarkers bottom-up data-dependent acquisition (DDA) mass spectrometry-based approaches are most popular. Drawbacks of this method are stochastic and irreproducible precursor ion selection. Recently, data-independent acquisition (DIA) emerged as an alternative method. It overcomes several limitations of DDA, since it combines the benefits of DDA and targeted methods like selected reaction monitoring (SRM). We established a DIA method for in-depth proteome analysis of CSF. For this, four spectral libraries were generated with samples from native CSF ( n = 5), CSF fractionation (15 in total) and substantia nigra fractionation (54 in total) and applied to three CSF DIA replicates. The DDA and DIA methods for CSF were conducted with the same nanoLC parameters using a 180 min gradient. Compared to a conventional DDA method, our DIA approach increased the number of identified protein groups from 648 identifications in DDA to 1574 in DIA using a comprehensive spectral library generated with DDA measurements from five native CSF and 54 substantia nigra fractions. We also could show that a sample specific spectral library generated from native CSF only increased the identification reproducibility from three DIA replicates to 90% (77% with a DDA method). Moreover, by utilizing a substantia nigra specific spectral library for CSF DIA, over 60 brain-originated proteins could be identified compared to only 11 with DDA. In conclusion, the here presented optimized DIA method substantially outperforms DDA and could develop into a powerful tool for biomarker discovery in CSF. Data are available via ProteomeXchange with the identifiers PXD010698, PXD010708, PXD010690, PXD010705, and PXD009624.

Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.

Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization.

New insight into stimulus-induced plasticity of the olfactory epithelium in Mus musculus by quantitative proteomics.

The olfactory system is exposed to a plethora of chemical compounds throughout an organism's lifespan. Anticipation of stimuli and construction of appropriate neural filters present a significant challenge. This may be addressed via modulation of the protein composition of the sensory epithelium in response to environmental conditions. To reveal the mechanisms governing these changes, we employed a comprehensive quantitative proteomics strategy. Two groups of juvenile mice were treated with either pulsed or continuous application of octanal. After 20 days of treatment, we performed a behavioral study and conducted electrophysiological recordings from the olfactory epithelium (OE). Both treated groups demonstrated peripheral desensitization to octanal; however, only the 'continuous' group exhibited habituation. To obtain novel insight into the molecular mechanisms underpinning the peripheral desensitization to octanal, the OE proteomes of octanal-treated mice versus control were quantitatively analyzed using two-dimensional difference gel electrophoresis. We identified several significantly regulated proteins that were functionally classified as calcium-binding proteins, cytoskeletal proteins, and lipocalins. The calcium-binding proteins and cytoskeletal proteins were up-regulated in the 'pulsed' group, whereas in the 'continuous' group, four lipocalins were significantly down-regulated. Uniquely, the lipocalin odorant-binding protein Ia was drastically down-regulated in both groups. The identified proteins reflect changes throughout the entire OE, corresponding to changes in neuronal, non-neuronal, and pericellular processes. We report the regulation of several promising candidates for the investigation of odorant-induced changes of the OE. Among these proteins are different lipocalins, which seem to play a crucial role in the regulation of the sensitivity of the olfactory system.

Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery.

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Delta mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.