Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines.

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.

In vivo trapping of FtsH substrates by label-free quantitative proteomics.

FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.

The membrane proteome of sensory cilia to the depth of olfactory receptors.

In the nasal cavity, the nonmotile cilium of olfactory sensory neurons (OSNs) constitutes the chemosensory interface between the ambient environment and the brain. The unique sensory organelle facilitates odor detection for which it includes all necessary components of initial and downstream olfactory signal transduction. In addition to its function in olfaction, a more universal role in modulating different signaling pathways is implicated, for example, in neurogenesis, apoptosis, and neural regeneration. To further extend our knowledge about this multifunctional signaling organelle, it is of high importance to establish a most detailed proteome map of the ciliary membrane compartment down to the level of transmembrane receptors. We detached cilia from mouse olfactory epithelia via Ca(2+)/K(+) shock followed by the enrichment of ciliary membrane proteins at alkaline pH, and we identified a total of 4,403 proteins by gel-based and gel-free methods in conjunction with high resolution LC/MS. This study is the first to report the detection of 62 native olfactory receptor proteins and to provide evidence for their heterogeneous expression at the protein level. Quantitative data evaluation revealed four ciliary membrane-associated candidate proteins (the annexins ANXA1, ANXA2, ANXA5, and S100A5) with a suggested function in the regulation of olfactory signal transduction, and their presence in ciliary structures was confirmed by immunohistochemistry. Moreover, we corroborated the ciliary localization of the potassium-dependent Na(+)/Ca(2+) exchanger (NCKX) 4 and the plasma membrane Ca(2+)-ATPase 1 (PMCA1) involved in olfactory signal termination, and we detected for the first time NCKX2 in olfactory cilia. Through comparison with transcriptome data specific for mature, ciliated OSNs, we finally delineated the membrane ciliome of OSNs. The membrane proteome of olfactory cilia established here is the most complete today, thus allowing us to pave new avenues for the study of diverse molecular functions and signaling pathways in and out of olfactory cilia and thus to advance our understanding of the biology of sensory organelles in general.

Identification of novel biomarker candidates for the immunohistochemical diagnosis of cholangiocellular carcinoma.

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.

Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines.

Within the past decade numerous methods for quantitative proteome analysis have been developed of which all exhibit particular advantages and disadvantages. Here, we present the results of a study aiming for a comprehensive comparison of ion-intensity based label-free proteomics and two label-based approaches using isobaric tags incorporated at the peptide and protein levels, respectively. As model system for our quantitative analysis we used the three hepatoma cell lines HepG2, Hep3B and SK-Hep-1. Four biological replicates of each cell line were quantitatively analyzed using an RPLC-MS/MS setup. Each quantification experiment was performed twice to determine technical variances of the different quantification techniques. We were able to show that the label-free approach by far outperforms both TMT methods regarding proteome coverage, as up to threefold more proteins were reproducibly identified in replicate measurements. Furthermore, we could demonstrate that all three methods show comparable reproducibility concerning protein quantification, but slightly differ in terms of accuracy. Here, label-free was found to be less accurate than both TMT approaches. It was also observed that the introduction of TMT labels at the protein level reduces the effect of underestimation of protein ratios, which is commonly monitored in case of TMT peptide labeling. Previously reported differences in protein expression between the particular cell lines were furthermore reproduced, which confirms the applicability of each investigated quantification method to study proteomic differences in such biological systems. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2).

The asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. In addition to HIF-α, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, to date, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD-containing substrate. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity through interaction with partitioning defective 3 homolog (Par-3). Using mass spectrometry we identified ASPP2 as a new substrate of FIH-1 but inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of FIH-1. However, FIH-1 depletion did lead to impaired binding of Par-3 to ASPP2 while the interaction between ASPP2 and p53, apoptosis and proliferation of the cancer cells were not affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in relocation of ASPP2 from cell-cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD-containing substrates can be modified by FIH-1-dependent hydroxylation. The large cellular pool of ARD-containing proteins suggests that FIH-1 can affect a broad range of cellular functions and signalling pathways under certain conditions, for example, in response to severe hypoxia.

Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets.

BACKGROUND: Platelets (PLTs) in stored PLT concentrates (PLCs) release PLT extracellular vesicles (PL-EVs) induced by senescence and activation, resembling the PLT storage lesion. No comprehensive classification or molecular characterization of senescence-induced PL-EVs exists to understand PL-EV heterogeneity. STUDY DESIGN AND METHODS: PL-EVs from 5-day-stored PLCs from healthy individuals were isolated and subfractionated by differential centrifugation, filtration, and density gradient ultracentrifugation into five PLT microvesicle (PL-MV) subfractions (Fraction [F]1-F5) and PLT exosomes (PL-EXs). PL-EV size, concentration, and composition were analyzed by nanoparticle tracking analysis, flow cytometry, and lipid and protein mass spectrometry. Protein data were verified by Western blot. RESULTS: PL-EVs showed overlapping mean particle sizes of 180 to 260 nm, but differed significantly in composition. Less dense, intermediate, and dense PL-MVs enriched specific lipidomic and proteomic markers related to the plasma membrane, intracellular membranes, PLT granules, mitochondria, and PLT activation. α-Synuclein (81% of total) accumulated in F1 and F2, amyloid-ß (Aß) precursor protein in F3 and F4 (84%), and apolipoprotein (Apo)E (88%) and ApoJ (92%) in F3 to F5. PL-EXs enriched lipid species and proteins, with high abundance of lipid raft, PLT adhesion, and immune response-related markers. CONCLUSION: Differential lipid and protein compositions of PL-EVs suggest their unique cellular origins and functions, partly overlapping with PLT granule secretion. Dense PL-MVs might represent autophagic vesicles released during PLT activation and apoptosis and PL-EXs resemble lipid rafts, with a potential role in PLT aggregation and immunity. Segregation of α-synuclein and Aß precursor protein, ApoE, and ApoJ into less dense and dense PL-MVs, respectively, show their differential carrier role of neurologic disease-related cargo.

Proteomic differences between hepatocellular carcinoma and nontumorous liver tissue investigated by a combined gel-based and label-free quantitative proteomics study.

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

Developing new methods to answer old and new questions in neurodegenerative diseases: 21(st) Workshop of the HUPO Brain Proteome Project (HBPP) 23-24 January 2014, Honolulu, Hawaii.

The HUPO Brain Proteome Project (HUPO BPP) held its 21(st) workshop in Honolulu, Hawaii. During the 23-24 January 2014 the island became the center of the open workshop of the scientific community.

Intestinal amino acid availability via PEPT-1 affects TORC1/2 signaling and the unfolded protein response.

The intestinal peptide transporter PEPT-1 plays an important role in development, growth, reproduction, and stress tolerance in Caenorhabditis elegans, as revealed by the severe phenotype of the pept-1-deficient strain. The reduced number of offspring and increased stress resistance were shown to result from changes in the insulin/IGF-signaling cascade. To further elucidate the regulatory network behind the phenotypic alterations in PEPT1-deficient animals, a quantitative proteome analysis combined with transcriptome profiling was applied. Various target genes of XBP-1, the major mediator of the unfolded protein response, were found to be downregulated at the mRNA and protein levels, accompanied by a reduction of spliced xbp-1 mRNA. Proteome analysis also revealed a markedly reduced content of numerous ribosomal proteins. This was associated with a reduction in the protein synthesis rate in pept-1 C. elegans, a process that is strictly regulated by the TOR (target of rapamycine) complex, the cellular sensor for free amino acids. These data argue for a central role of PEPT-1 in cellular amino acid homeostasis. In PEPT-1 deficiency, amino acid levels dropped systematically, leading to alterations in protein synthesis and in the IRE-1/XBP-1 pathway.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

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.

Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress.

Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.

Genome-wide characterization of miR-34a induced changes in protein and mRNA expression by a combined pulsed SILAC and microarray analysis.

The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR-34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. ∼19% of the detected proteins were differentially regulated, with 113 proteins being down- and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3'-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e.g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seed-match. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR-34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins.

Dynamic changes of the Caenorhabditis elegans proteome during ontogenesis assessed by quantitative analysis with 15N metabolic labeling.

The development of the nematode Caenorhabditis elegans is a highly dynamic process. Although various studies have assessed global transcriptome changes, information on the dynamics of the proteome during ontogenesis is not available. We metabolically labeled C. elegans by using ¹5N ammonium chloride as a precursor in Escherichia coli feeding bacteria grown in minimal media as a new cost-effective technique. Quantitative proteome analysis was performed by LC-MS/MS in animals harvested at different times during ontogenesis. We identified and quantified 245 proteins at all larval stages in two independent replicates. Between larval stages (20 and 40 h after hatching) 61 were found to change significantly in level. Among those ribosomal proteins, aminoacyl tRNA synthetases and enzymes of energy metabolism increased in abundance, while extracellular matrix proteins and muscle proteins dominated groups displaying reduced levels. Moreover, changes observed for selected proteins such as VIT-6 and SOD-1 matched with previously published findings confirming the validity of our approach. The metabolic labeling technique applied seems well suited to assess changes in the proteome changes of C. elegans in a quantitative manner during larval development. The data set generated provides the basis for further exploitation of the role of individual proteins or protein clusters during ontogenesis.

Identification of core components and transient interactors of the peroxisomal importomer by dual-track stable isotope labeling with amino acids in cell culture analysis.

The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.

Top-down de Novo protein sequencing of a 13.6 kDa camelid single heavy chain antibody by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry.

The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T(3)-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight >13 kDa has been established by mass spectrometric top-down sequencing.

Proteolipid protein is required for transport of sirtuin 2 into CNS myelin.

Mice lacking the expression of proteolipid protein (PLP)/DM20 in oligodendrocytes provide a genuine model for spastic paraplegia (SPG-2). Their axons are well myelinated but exhibit impaired axonal transport and progressive degeneration, which is difficult to attribute to the absence of a single myelin protein. We hypothesized that secondary molecular changes in PLP(null) myelin contribute to the loss of PLP/DM20-dependent neuroprotection and provide more insight into glia-axonal interactions in this disease model. By gel-based proteome analysis, we identified >160 proteins in purified myelin membranes, which allowed us to systematically monitor the CNS myelin proteome of adult PLP(null) mice, before the onset of disease. We identified three proteins of the septin family to be reduced in abundance, but the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 2 (SIRT2) was virtually absent. SIRT2 is expressed throughout the oligodendrocyte lineage, and immunoelectron microscopy revealed its association with myelin. Loss of SIRT2 in PLP(null) was posttranscriptional, suggesting that PLP/DM20 is required for its transport into the myelin compartment. Because normal SIRT2 activity is controlled by the NAD+/NADH ratio, its function may be coupled to the axo-glial metabolism and the long-term support of axons by oligodendrocytes.

Next-Generation Trapping of Protease Substrates by Label-Free Proteomics.

AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.

Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies.

Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here, we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs, "plastic antibodies") featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells, human neuroblastoma SH-SY5Y cells, mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography, pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate, exceeding the number identified by TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites.