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Listeria monocytogenesis a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine againstListeriais available. AttenuatedListeriastrains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screened forListeriaepitopes presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detected 68Listeriaepitopes from 42 different bacterial proteins, including several known antigens. Peptide epitopes presented on different cell lines were often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations resulted in specific CD8+ T-cell responses and high levels of protection in vaccination challenge experiments in mice. Our results pave the way for the development of a clinical mRNA vaccine againstListeriaand aid to improve attenuatedListeriavaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

Ribosome profiling has revealed translation outside canonical coding sequences, including translation of short upstream ORFs, long noncoding RNAs, overlapping ORFs, ORFs in UTRs, or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling, and CRISPR-based screens showed that hundreds of ORFs derived from noncoding transcripts produce (micro)proteins, whereas other studies failed to find evidence for such types of noncanonical translation products. Here, we attempted to discover translation products from noncoding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. We show that, theoretically our strategy facilitates the detection of translation events of transcripts from non -coding regions but experimentally only find 19 peptides that might originate from such translation events. Finally, Virotrap-based interactome analysis of two N-terminal proteoforms originating from noncoding regions showed the functional potential of these novel proteins.

Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. Virotrap based interactome analysis of two N-terminal proteoforms originating from non-coding regions finally showed the functional potential of these novel proteins.

Surface enhanced Raman spectroscopy (SERS) is a selective and sensitive technique, which allows for the detection of protease activity by monitoring the cleavage of peptide substrates. Commonly used free-space based SERS substrates, however, require the use of bulky and expensive instrumentation, limiting their use to laboratory environments. An integrated photonics approach aims to implement various free-space optical components to a reliable, mass-reproducible and cheap photonic chip. We here demonstrate integrated SERS detection of trypsin activity using a nanoplasmonic slot waveguide as a waveguide-based SERS substrate. Despite the continuously improving SERS performance of the waveguide-based SERS substrates, they currently still do not reach the SERS enhancements of free-space substrates. To mitigate this, we developed an improved peptide substrate in which we incorporated the non-natural aromatic amino acid 4-cyano-phenylalanine, which provides a high intrinsic SERS signal. The use of non-natural aromatics is expected to extend the possibilities for multiplexing measurements, where the activity of several proteases can be detected simultaneously.

We used a nanoplasmonic slot waveguide for the first time demonstration of waveguide-based detection of protease activity using Surface-Enhanced Raman Spectroscopy (SERS), paving the way for an integrated lab-on-a-chip protease activity monitoring.

Surface Enhanced Raman Spectroscopy (SERS) allows sensitive and selective detection of protease activity by monitoring the cleavage of specific peptide substrates. Furthermore, it offers the possibility for multiplexing, during which the activity of two (or more) proteases with different specificities is detected simultaneously. To distinguish between the contributions of different proteases, different aromatic amino acids with non-overlapping SERS peaks need to be used as Raman reporters. As the three natural aromatic amino acids only offer limited possibilities for multiplexing, we examined several non-natural aromatic amino acids with the aim of expanding multiplexing possibilities. We recorded their SERS spectra for the Raman shifts of 300-1700 cm–1 and identified their characteristic SERS peaks. Of the examined nonnatural aromatic amino acids, 3-nitro-tyrosine and two phenylalanines containing stable heavy isotopes seem particularly promising for multiplexing applications. Besides exhibiting characteristic SERS peaks in the spectral region of interest, these non-natural aromatics provide strong SERS peaks compared to natural aromatic amino acids, consequently improving detection sensitivity.

Surface-enhanced Raman scattering provides a promising technology for sensitive and selective detection of protease activity by monitoring peptide cleavage. Not only are peptides and plasmonic hotspots similarly sized, Raman fingerprints also hold large potential for spectral multiplexing. Here, we use a gold-nanodome platform for real-time detection of trypsin activity on a CALNNYGGGGVRGNF substrate peptide. First, we investigate the spectral changes upon cleavage through the SERS signal of liquid-chromatography separated products. Next, we show that similar patterns are detected upon digesting surface-bound peptides. We demonstrate that the relative intensity of the fingerprints from aromatic amino acids before and after the cleavage site provides a robust figure of merit for the turnover rate. The presented method offers a generic approach for measuring protease activity, which is illustrated by developing an analogous substrate for endoproteinase Glu-C.

Surface-Enhanced Raman Spectroscopy (SERS) allows for the highly specific detection of molecules by enhancing the inherently weak Raman signals near the surface of plasmonic nanostructures. A variety of plasmonic nanostructures have been developed for SERS signal excitation and collection in a conventional free-space microscope, among which the gold nanodomes offer one of the highest SERS enhancements. Nanophotonic waveguides have recently emerged as an alternative to the conventional Raman microscope as they can be used to efficiently excite and collect Raman signals. Integration of plasmonic structures on nanophotonic waveguides enables reproducible waveguide-based excitation and collection of SERS spectra, such as in nanoplasmonic slot waveguides. In this paper, we compare the SERS performance of gold nanodomes, in which the signal is excited and collected in free space, and waveguide-based nanoplasmonic slot waveguide. We evaluate the SERS signal enhancement and the SERS background of the different SERS platforms using a monolayer of nitrothiophenol. We show that the nanoplasmonic slot waveguide approaches the gold nanodomes in terms of the signal-to-background ratio. We additionally demonstrate the first-time detection of a peptide monolayer on a waveguide-based SERS platform, paving the way towards the SERS monitoring of biologically relevant molecules on an integrated lab-on-a-chip platform.

Highlight We identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots., Roots explore the soil for water and nutrients through the continuous production of lateral roots. Lateral roots are formed at regular distances in a steadily elongating organ, but how future sites for lateral root formation become established is not yet understood. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots. In addition, based on genetic and expression data, we found evidence for the involvement of its proposed receptor, XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1), during the process of lateral root initiation. In conclusion, we report here on the existence of a peptide ligand−receptor kinase interaction that impacts lateral root initiation. Our results represent an important step towards the understanding of the cellular communication implicated in the early phases of lateral root formation.

In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

Gelsolin is an actin-binding protein (82 kDa) consisting of six repeated segments (S1-S6), each approximately 120 residues long. It interacts with phospholipids and we previously showed that phosphatidylinositol 4,5-bisphosphate promotes phosphorylation of gelsolin by the tyrosine kinase c-Src. We used a combination of different methods, such as thin-layer chromatography and anti-phosphotyrosine-agarose immunoprecipitation of phosphopeptides combined with matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and post source decay (PSD) analysis, to identify the phosphorylation sites in gelsolin. The major phosphorylation site (Tyr438) was located in subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin-actin2 complex was inhibited by 90%. Gelsolin phosphorylation by c-Src in the presence of lysophosphatidic acid also revealed Tyr438 as the most prominent site. Additional minor sites were found using the anti-phosphotyrosine bead immunoprecipitation method followed by MALDI-MS and PSD analysis. These sites, representing approximately 5% of the total phosphate incorporation, were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we generated antibodies which specifically recognize Tyr438 phosphorylated gelsolin.

We analysed the involvement of proteases during taxol-mediated cell death of human A549 non-small-cell lung carcinoma cells using a proteomics approach that specifically targets protein N termini and further detects newly formed N termini that are the result of protein processing. Our analysis revealed 27 protease-mediated cleavages, which we divided in sites C-terminal to aspartic acid (Asp) and sites C-terminal to non-Asp residues, as the result of caspase and non-caspase protease activities, respectively. Remarkably, some of the former were insensitive to potent pancaspase inhibitors, and we therefore suggest that previous inhibitor-based studies that report on the caspase-independent nature of taxol-induced cell death should be judged with care. Furthermore, many of the sites C-terminal to non-Asp residues were also uniquely observed in a model of cytotoxic granule-mediated cell death and/or found by in vitro cataloging human mu-calpain substrates using a similar proteomics technique. This thus raises the hypothesis that killing tumor cells by chemotherapy or by immune cells holds similar non-Asp-specific proteolytic components with strong indications to calpain activity.

A new approach for proteome-wide analysis of sialylated N-glycopeptides based on the diagonal chromatographic COFRADIC technology is presented here. The use of α(2−3,6,8,9) neuraminidase is central to isolate sialylated N-glycopeptides out of a complex peptide mixture. Two different COFRADIC techniques are introduced here, either without or with post-metabolic oxygen-18 labeling (direct versus indirect sorting), and when applied to immuno-depleted mouse serum, we herewith identified 93 sialylated glycosylation sites in 53 serum proteins. Keywords: diagonal chromatography • sialic acid • serum • COFRADIC

Reactive oxygen species such as hydrogen peroxide can modify proteins via direct oxidation of their sulfur-containing amino acids, cysteine and methionine. Methionine oxidation, studied here, is a reversible posttranslational modification that is emerging as a mechanism by which proteins perceive oxidative stress and function in redox signaling. Identification of proteins with oxidized methionines is the first prerequisite toward understanding the functional effect of methionine oxidation on proteins and the biological processes in which they are involved. Here, we describe a proteome-wide study of in vivo protein-bound methionine oxidation in plants upon oxidative stress using Arabidopsis thaliana catalase 2 knock-out plants as a model system. We identified over 500 sites of oxidation in about 400 proteins and quantified the differences in oxidation between wild-type and catalase 2 knock-out plants. We show that the activity of two plant-specific glutathione S-transferases, GSTF9 and GSTT23, is significantly reduced upon oxidation. And, by sampling over time, we mapped the dynamics of methionine oxidation and gained new insights into this complex and dynamic landscape of a part of the plant proteome that is sculpted by oxidative stress. ispartof: Molecular & Cellular Proteomics vol:14 issue:5 pages:1217-1229 ispartof: location:United States status: published

The physiological roles of the granzymes A and K have been debated, especially concerning their involvement in cytotoxic and inflammatory processes. By performing N-terminal COFRADIC assisted N-terminomics on the homologous human granzymes A and K, we here provide detailed data on their substrate repertoires, their specificities, and differences in efficiency by which they cleave their substrates, all of which may aid in elucidating their key substrates. In addition, the so far uncharacterized mouse granzyme K was profiled alongside its human orthologue. While the global primary specificity profiles of these granzymes appear quite similar as they revealed only subtle differences and pointed to substrate occupancies in the P1, P1', and P2' position as the main determinants for substrate recognition, differential analyses unveiled distinguishing substrate subsite features, some of which were confirmed by the more selective cleavage of specifically designed probes.

We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+-immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O-18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190 phosphorylated peptides from 152 different proteins. This dataset includes 38 novel protein phosphorylation sites.

Peritoneal endometriosis is the result of ectopic implantation and growth of endometrium tissue that has been regurgitated into the abdominal cavity during menstruation. We have previously shown that menstrual effluent induces epithelial to mesenchymal transitions (EMT) in mesothelial cells, which results in cell retraction and exposure of submesothelial extracellular matrix. Since endometrial tissue preferentially adheres to the extracellular matrix, adhesion of endometrial tissue to the peritoneum is facilitated. The EMT were shown to be associated with differential expression and phosphorylation of mesothelial proteins. Using radiolabeling and proteomics we detected changes in protein expression and phosphorylation that occur in mesothelial cells during the EMT process. The identity of 73 proteins, which were obtained from 324 analyzed spots, was confirmed. The expression of 35 proteins involved in organization of the cytoskeleton, signal transduction, regulation of the redox state, and production of ATP, was altered during the EMT process. Four of the identified proteins were differentially phosphorylated: annexin-1, an actin-binding protein and a substrate for receptor tyrosine kinases; tropomyosin-alpha, a regulator of actin filament stability and cell shape; elongation factor 1 delta; ATP synthase beta-chain. In conclusion, factors from menstrual effluent induce specific changes in the expression and phosphorylation status of structural, regulatory and metabolic proteins relevant to the complex process of EMT in mesothelial cells.

We describe a protocol for quantitative labeling of tryptic peptides with oxygen-18. Proteins are first digested in natural water with trypsin, the pH is then lowered to 4.5 and the mixture is dried. Oxygen-18 water is added and two oxygen-18 atoms are incorporated at the peptides' carboxyl termini. Trypsin is finally inactivated by cysteine alkylation under denaturing conditions, which blocks oxygen back-exchange. The general value of this labeling strategy for differential proteomics is illustrated by the analysis and identification of several couples of differently labeled amino terminal peptides isolated from a human platelet proteome by a previously described chromatographic procedure. Keywords: nongel proteomics • mass spectrometry • oxygen-18 • differential analysis • COFRADIC

A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of two housekeeping enzymes. The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2-day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S-adenosylmethionine synthetase that catalyses the synthesis of S-adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different pI and Mr. Both proteins were absent in dry mature seeds and in 1-day imbibed seeds, but specifically accumulated at the moment of radicle protrusion. Arabidopsis seed germination was strongly delayed in the presence of dl-propargylglycine, a specific inhibitor of methionine synthesis. Furthermore, this compound totally inhibited seedling growth. These phenotypic effects were largely alleviated upon methionine supplementation in the germination medium. The results indicated that methionine synthase and S-adenosylmethionine synthetase are fundamental components controlling metabolism in the transition from a quiescent to a highly active state during seed germination. Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in Arabidopsis only after radicle protrusion.

Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with35S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitin-conjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochromecoxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.

A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of l -galactosyl residues, catalyzed by a largely unknown GDP- d -mannose 3",5"-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP- d -mannose 3",5"-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a blast search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP- d -mannose 5"-epimerase. The plant GDP- d -mannose 3",5"-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

We here describe the use of added reversed-phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide-bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix-assisted laser desorption/ionization (MALDI)-matrix containing organic/aqueous solvent and transferred to a MALDI-target for mass analysis. We show that by using this bead-peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 microL. We have used this concentration procedure in combination with MALDI-post-source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead-peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI-mass spectrometry (MS) experiments. We have found additionally that the bead-peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead-peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI-MS sample preparation process.

We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse-phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization-reflectron time of flight (MALDI-RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide-matrix mixtures. This permits high yield identification and sequence tagging by post-source-decay analysis on peptides derived from proteins only available in the femtomole range from one-dimensional (1-D) or two-dimensional (2-D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.

As part of our study of isoenzyme forms of human cytochrome c oxidase, we purified subunit IV from human heart and skeletal muscle with reversed-phase HPLC and determined the N-terminal amino acid sequences and the electrophoretic mobility. The N-terminus of human heart subunit IV proved to be ragged with 30% of the protein lacking the first three residues. Also a Tyr/Phe polymorphism was observed at residue 16. No differences in N-terminal sequence and electrophoretic mobility were observed between subunit IV of cytochrome c oxidase from human heart and skeletal muscle. Therefore, our results suggest that identical subunits IV are present in cytochrome c oxidase from human heart and skeletal muscle. A putative isoform of subunit IV with a blocked N-terminus was purified from human heart cytochrome c oxidase, which proved to have a different retention time on a reversed-phase column and also a slightly higher electrophoretic mobility on an SDS-polyacrylamide gel compared to the native subunit IV. We could not demonstrate the existence of isoforms of subunit IV in human skeletal muscle.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.

International audience; Actin-based comet tails produced by Listeria monocytogenes are considered as representative models for cellular force-producing machineries crucial for cell migration. We here present a proteomic picture of these tails formed in extracts from brain and platelets. This provides a comprehensive view, revealing high molecular complexity and novel host cell proteins as tail components, and suggests the participation of specific multicomponent regulatory complexes. This work forms a new basis to expand current models of cellular protrusion.

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.

Top of pageAbstract Co-chaperone p23 is a component of the heat-shock protein (Hsp)90 multiprotein-complex and is an important modulator of Hsp90 activity. Hsp90 client proteins involved in oncogenic survival signaling are frequently mutated in leukemia, and the integrity of the Hsp90 complex could therefore be important for leukemic cell survival. We demonstrate here that p23 is cleaved to a stable 17 kDa fragment in leukemic cell lines treated with commonly used chemotherapeutic drugs. The cleavage of p23 paralleled the activation of procaspase-7 and -3 and was suppressed by the caspase-3/-7 inhibitor DEVD-FMK. In vitro translated 35S-p23 (in reticulocyte lysate) was cleaved at D142 and D145 by caspase-7 and -3. Cleavage of p23 occurred in caspase-3-deficient MCF-7 cells, suggesting a role for caspase-7 in intact cells. The Hsp90 inhibitor geldanamycin enhanced caspase-dependent p23 cleavage both in vitro and in intact cells. Geldanamycin also enhanced anthracycline-induced caspase activation and apoptosis. We conclude that p23 is a prominent target in leukemic cell apoptosis. Geldanamycin enhanced p23 cleavage both by rendering p23 more susceptible to caspases and by enhancing chemotherapy-induced caspase activation. These findings underscore the importance of the Hsp90-complex in antileukemic treatment, and suggest that p23 may have a role in survival signaling. Keywords: AML, anthracyclines, co-chaperone p23, geldanamycin, caspase-7

Hypoxia-inducible factor-1 (HIF) is regulated by oxygen-dependent prolyl hydroxylation. Of the three HIF prolyl hydroxylases (PHD1, 2 and 3) identified, PHD3 exhibits restricted substrate specificity in vitro and is induced in different cell types by diverse stimuli. PHD3 may therefore provide an interface between oxygen sensing and other signalling pathways. We have used co-purification and mass spectrometry to identify proteins that interact with PHD3. The cytosolic chaperonin TRiC was found to copurify with PHD3 in extracts from several cell types. Our results indicate that PHD3 is a TRiC substrate, providing another step at which PHD3 activity may be regulated.

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments

A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component α-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for thega1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a β-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) are both substrates of the 43-kDa type I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensitive inhibition by 70-85% of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) 5-phosphatase activities was observed in homogenates from rat cortical astrocytes, human astrocytoma 1321N1 cells, and rat basophilic leukemia RBL-2H3 cells after incubation with carbachol. The effect was reproduced in response to UTP in rat astrocytic cells and Chinese hamster ovary cells overexpressing human type I 5-phosphatase. Immunodetection as well as mass spectrometric peptide mass fingerprinting and post-source decay (PSD) sequence data analysis after immunoprecipitation permitted unambiguous identification of the major native 5-phosphatase isoform hydrolyzing Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) as type I inositol polyphosphate 5-phosphatase. In ortho-(32)P-preincubated cells, the phosphorylated 43 kDa-enzyme could be identified after receptor activation by immunoprecipitation followed by electrophoretic separation. Phosphorylation of type I 5-phosphatase was blocked after cell preincubation in the presence of Ca(2+)/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62). In vitro phosphorylation of recombinant type I enzyme by Ca(2+)/calmodulin kinase II resulted in an inhibition (i.e. 60-80%) of 5-phosphatase activity. In this study, we demonstrated for the first time a novel regulation mechanism of type I 5-phosphatase by phosphorylation in intact cells.

Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.

Due to its very short analysis time, its high sensitivity and ease of automation, matrix-assisted laser desorption/ionization (MALDI)-peptide mass fingerprinting has become the preferred method for identifying proteins of which the sequences are available in databases. However, many protein samples cannot be unambiguously identified by exclusively using their peptide mass fingerprints (e.g., protein mixtures, heavily posttranslationally modified proteins and small proteins). In these cases, additional sequence information is needed and one of the obvious choices when working with MALDI-mass spectrometry (MS) is to choose for post source decay (PSD) analysis on selected peptides. This can be performed on the same sample which is used for peptide mass fingerprinting. Although in this type of peptide analysis, fragmentation yields are very low and PSD spectra are often very difficult to interpret manually, we here report upon our five years of experience with the use of PSD spectra for protein identification in sequence (protein or expressed sequence tag (EST)) databases. The combination of peptide mass fingerprinting and PSD and analysis described here generally leads to unambiguous protein identification in the amount of material range generally encountered in most proteome studies.

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments.

Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.

The complete amino acid sequence of the low-redox potential cytochrome c-551.5 from Rhodobacter sphaeroides was determined by automated Edman degradation combined with mass spectroscopy. There are 139 residues and two typical Cys-X-X-Cys-His heme-binding sites. A homologous low-redox potential cytochrome was also sequenced from Rhodobacter adriaticus and was found to contain 126 residues. It is 53% identical to that of Rb. sphaeroides and has two internal deletions of one and five residues. The Rhodobacter diheme cytochromes are 21-24% identical to the translated open reading frame SLL1886 from Synechocystis sp. PCC6801. There are at least two deletions of five and eight residues in the 188-residue cyanobacterial protein. Each of the three cytochromes has more histidines than it needs to bind the two hemes, but conserved histidines located 23 residues after the first heme and 14-19 residues before the second heme are likely to be the sixth heme ligands. There is no evidence for gene doubling and no similarity to any other known cytochromes. The measured helix content of 24% is much less than normal for c-type cytochromes. These proteins thus appear to be representative of an entirely new class of c-type cytochromes.