Search

A novel interface adapter has been designed to provide a new way of directly coupling a nano-liquid chromatograph to an electron ionization mass spectrometer. It connects the transfer capillary coming from the liquid chromatograph to the ionization chamber and can be easily screwed into the ion source. Liquid coming from the column passes through the heated adapter flow path and is vaporized. A continuous flow of new liquid pushes the vapor into the ionization chamber where it is ionized and continues on to the mass analyzer. The advantages of the new adapter are reduced ice formation inside the ion source and less clogging of the transfer capillary. Improvements achieved are demonstrated on the basis of caffeine and steroid analysis. The limits of detection of selected steroids are compared with and without the adapter. The adapter improves the detection limit of the system by a factor of 2 and precision from ≤15% to ≤9% relative standard deviation. No derivatization procedure is necessary before the analysis of small polar compounds. The resulting spectra are reproducible, easily interpretable, and database searchable. The new method is robust, delivers reproducible results, and provides a highly efficient alternative to existing methods in the field of pharmaceutical analysis. Copyright © 2011 John Wiley & Sons, Ltd.

A simple, sensitive and fast method for the determination of melamine and its derivatives in milk powder using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed. Neither time-consuming sample preparation, nor special target plates, or other extra equipment are necessary. The common matrix sinapinic acid (SA) was used with a dried-droplet preparation. Detection limits (signal-to-noise (S/N) ratio = 3) for standard solutions of melamine, ammeline and cyanuric acid were 10, 25 and 10 µg/L, respectively. The limit of quantification (LOQ) for melamine was 25 µg/L and excellent linearity (R2: 0.9990) was maintained over the range of 10–2000 µg/L. Ammeline and cyanuric acid were analyzed with an LOQ of 50 µg/L and also excellent linearity (R2: 0.9997 and R2: 0.9998). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of melamine, ammeline and cyanuric acid in milk powder samples with a simple sample preparation. The LOQ of melamine was 0.5 µg/g. Ammeline and cyanuric acid were detectable at 0.5 and 5 µg/g. This method showed excellent accuracy, precision and linearity and significantly reduces the needed analysis time, as only approximately 10 s/sample measuring time is required. To the authors' knowledge, this is the first published method to quantify melamine and derivatives by MALDI-TOF-MS. Copyright © 2011 John Wiley & Sons, Ltd.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a highly appreciated method in oligosaccharide analysis due to its high sensitivity and ease of use. As underivatized oligosaccharides suffer from low ionization efficiency, derivatization is a widespread tool. However, subsequent sample purification and toxic or otherwise dangerous reagents complicate the oligosaccharides’ analysis. On-target derivatization performed by the matrix 3-aminoquinoline does not require such purification and yields Schiff bases which can be measured in positive and negative ion modes from one single spot. In this article, this simple and convenient method is applied for structural characterization of oligosaccharides using a MALDI LTQ Orbitrap. Information received by the different fragmentation techniques collision-induced dissociation (CID), pulsed-Q dissociation (PQD) and higher energy C-trap dissociation (HCD) are compared. By combining the information received from fragmentation of [M+H] + ions (in positive ion mode) and [M+NO 3 ] − ions (in negative ion mode) of 3-AQ-derivatized species by all three fragmentation techniques, a complete structural characterization in terms of linkage, branching and anomeric configuration of glycosidic bonds of oligosaccharides could be achieved. The analysis of isomeric human milk oligosaccharides derivatized with 3-AQ yielded comprehensive information on the isomers’ structures. Finally, a simple combination of peak lists obtained by the different fragmentation techniques and automatic measurements enhance and facilitate oligosaccharide analysis.

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.

The final step of B-cell maturation is to differentiate into plasma cells, a process that is accompanied by gross changes in subcellular organization to enable antibody secretion. To better understand this critical step in mounting a humoral immune response, we analyzed proteome dynamics during plasma cell differentiation with combined 2-DE/MS. Thirty-two identified protein spots changed in relative abundance when lipopolysaccharide (LPS)-stimulated primary B cells differentiated into antibody-secreting plasma cells. A correlative analysis of protein and transcript abundance suggested that one third of these proteins are post-transcriptionally regulated. Apart from ER-resident chaperones, lipid metabolic enzymes, and translation initiation factors, we identified several proteins that had not been previously studied in plasma cells. Among them is the transiently upregulated proteasome activator (PA) 28γ, a component of the putative nuclear proteasome. Additionally, we discovered that the non-canonical inflammatory cytokine high-mobility group box 1 (HMG1) was released from plasma cells into the extracellular milieu. This suggests a novel role for plasma cells as pro-inflammatory mediators, which has important implications for various autoimmune diseases and chronic inflammation.

We investigate the conflict between authors and their publishers that may result from a copyright system that allows authors to transfer copyrights to an additional publisher. A two-period bargaining model analyzes effects of competition, time preferences, and bargaining power on license prices, publisher investments, and authors' lifetime incomes. We demonstrate that authors benefit from the new copyright system if new publishers continue the distribution of their orphaned works. Authors do not necessarily benefit if exclusive publishers are still exploiting licenses, because high levels of competition result in underinvestment by publishers and in internalization effects during contract negotiations.

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Psi). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Psi1191 methylation. ESI MS analysis of acp-modified Psi-nucleosides in a DeltaNep1-mutant showed that Nep1 catalyzes the Psi1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a deltasnr35 mutant. This strongly suggests a dual Nep1 function, as Psi1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Adipose tissue is an endocrine organ secreting different types of proteins, known as adipokines. These adipokines play important roles in homeostasis and metabolism. Adipocyte differentiation leads to a change in adipokine secretion profile which is probably involved in disruption of homeostasis. Many adipokines have been identified but species differences and limitations of human adipose tissue material urged the need for better model systems. Here we used a human cell strain derived from a Simpson Golabi Behmel syndrome (SGBS) patient. SGBS cells have already been used in functional studies on adipocytes but not in a proteomic search for adipokines. In this study, 2D-MS/MS and nLC-MALDI-MS/MS were applied to investigate secretion profiles of SGBS adipokines. A total of 80 secreted proteins were identified; 6 proteins are novel adipocyte secreted proteins, 20 proteins have not been detected before in human adipose material and 23 additional proteins previously detected in visceral adipose tissue have been found here secreted by SGBS-cells of subcutaneous origin. It can be concluded that SGBS cells are both a valid human cell model for adipocyte secretion profiling and for searching for novel human (pre)adipocytes secreted proteins.

This review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequence coverage, as the review title already implies, seems to be rather surreal if the complexity and dynamic range of a proteome is taken into consideration. Nevertheless, established bottom-up shotgun approaches are able to roughly identify a complete proteome as exemplary shown by yeast. However, this proceeding ignores more or less the fact that a protein is present as various protein species. The unambiguous identification of protein species requires 100% sequence coverage. Furthermore, the separation of the proteome must be performed on the protein species and not on the peptide level. Therefore, top-down is a good strategy for protein species analysis. Classical 2D-electrophoresis followed by an enzymatic or chemical cleavage, which is a combination of top-down and bottom-up, is another interesting approach. Moreover, the review summarizes further top-down and bottom-up combinations and to which extent middle-down improves the identification of protein species. The attention is also focused on cleavage strategies other than trypsin, as 100% sequence coverage in bottom-up experiments is only obtainable with a combination of cleavage reagents.

A new method, based on a direct-electron ionization (EI) interface, is presented for the analysis of compounds insufficiently amenable to usual MS methods. The instrumentation is composed of a nano liquid chromatograph (LC) and a mass spectrometer (MS) directly coupled by a transfer capillary. The eluent is directly introduced into the heated electron impact ion source of the MS. Significant advantages are the generation of reproducible spectra and the ability to ionize highly polar compounds. Boronic acids are used as coupling reagents to produce drugs, agrochemicals, or herbicides. The purity of educts is of high importance because impurities in the educt are directly associated with impurities in the product. Because of their high polarity and tendency to form boroxines, boronic acids require derivatization for GC analysis. The presented nano-LC-EI/MS method is easily applicable for a broad range of boronic acids. The method shows good detection limits for boronic acids up to 200 pg, is perfectly linear, and shows a very high robustness and reproducibility. A mixture of compounds could easily be separated on a monolithic RP18e column. The method represents a new, simple, robust, and reproducible approach for the detection of polar analytes. It is a good candidate to become a standard method for industrial applications.

Peptide Mass Fingerprinting (PMF) of tryptically in-gel digested samples is a well-established protein identification technique for MALDI mass spectrometry but an in-depth PMF evaluation for in-gel digestions of less specific enzymes is still missing. This study demonstrates that the MALDI-LTQ-Orbitrap provides the mass accuracy to gain significant database search results via PMF for the less specific enzymes chymotrypsin and elastase. Additionally, the highly sensitive MALDI matrix ClCCA was compared to the most widely used matrix CHCA by means of the detected peptide number, peptide composition, pI and S/N distribution, sequence coverage, and Mascot score. Therefore, several proteins were in-gel digested by chymotrypsin and elastase. Trypsin and proteinase K were included as references for specific and nonspecific proteases, respectively. Compared to CHCA, ClCCA resulted in a better mapping in all cases of the more complex peptide mixtures generated by less specific enzymes. In summary, the MALDI-LTQ-Orbitrap combined with the matrix ClCCA makes PMF of less specific digests possible in an easy and fast way. Moreover, it opens more possibilities for PMF in the analysis of difficult tasks such as membrane proteins.

Chlorobium tepidum is a Gram-negative bacterium of the green sulfur phylum (Chlorobia). Chlorobia are obligate anaerobic photolithoautotrophs that are widely distributed in aquatic environments where anoxic layers containing reduced sulfur compounds are exposed to light. The envelope of C. tepidum is a complex organelle composed of the outer membrane, the periplasm-peptidoglycan layer, and the cytoplasmic membrane. In addition to the outer and plasma membranes, C. tepidum contains chlorosomes attached to the cytoplasmic side of the plasma membrane. Each cellular compartment has a unique set of proteins, called sub-proteome. An important aim of proteome analysis is to study the level of the expressed genes and their response to environmental changes. Membrane protein studies are of primary importance to understand how nutrients are transported inside the cell, how toxic molecules are exported, and the mechanisms of photosynthesis and energy metabolism.

Dedicated and specific sample preparation and adequate chromatographic resolution prior to MS are necessary for comprehensive and site-specific glycosylation analysis to compensate for high heterogeneity of protein glycosylation, low-abundance of specific glycoforms and ion-suppression effects caused by coelution of other peptides. This article describes a scheme for glycopeptide profiling, which comprises HILIC batch enrichment followed by complementary HILIC and RP-LC in 1-D and 2-D approaches. For reproducible and sensitive nano-LC/ESI-MS analysis, we used ZIC-HILIC and RP18e monolithic silica capillaries and assessed their retention characteristics and complementarity for glycopeptide separations. The experiments revealed that pre-enrichment of glycopeptides in combination with LC employing both phases considerably improves site-specific elucidation of glycosylation heterogeneity. Zwitterionic hydrophilic interaction liquid chromatography showed high capability to separate glycopeptides by their glycan composition, which coeluted on RP18e. By varying solvent conditions, retention can be well tuned, and efficient separations were achieved even in absence of any additives like salt or formic acid. RP18e facilitated glycopeptide separations with high peak capacity based on peptide sequence and degree of sialylation. Implementing both orthogonal and complementary phases in 1-D and 2-D LC setups was shown to significantly increase the number of different identified glycoforms and possesses great potential for comprehensive glycoproteomics approaches.

Liquid chromatography, coupled with tandem mass spectrometry, is an established method for the identification of proteins from a complex sample. Despite its wide application, the analysis of whole proteomes still represents a challenge to researchers, because of the complexity and dynamic range of protein concentrations in biological samples. The analysis of such samples can be improved by adding a prefractionation step or a combination of orthogonal separation techniques. Off-gel isoelectric focusing (OGE) has successfully been used for prefractionation of a tryptic digest prior to nLC separation. In contrast to previous published results, we present a complete glycerol-free OGE for the analysis of purple membranes and Corynebacterium glutamicum membranes using the less-specific enzyme elastase. More than 85% of the identified unique peptides were found in solely one fraction, with very little carryover. These results are in accordance with those published for tryptic peptides. Therefore, OGE can be used as an effective prefractionation method in a multidimensional separation experiment of nontryptic membrane peptides.

SDS-PAGE is still one of the most widespread separation techniques in proteomic research and usually coupled to subsequent MS measurement for protein identification. The proteins are digested while embedded in the gel matrix. The resultant peptides are eluted out of the gel and finally analyzed. The in-gel digestion process suffers from several drawbacks which influence the experimental outcome with respect to protein sequence coverage and detection sensitivity. Limited accessibility of the protease to the substrate protein and insufficient peptide extraction represent the two major problems. To specifically target these issues, we established a novel partly reversible gel system, in which the gel matrix can be conditionally cleaved to increase the pore diameters. By using a crosslinker mixture consisting of Bis and ethylene-glycol-diacrylate the acrylamide filament interconnections can be partly hydrolyzed in alkaline solution. The new hybrid gels have been tested to be compatible with a variety of acidic staining techniques. They exhibit similar electrophoretic performance compared with regular solely Bis-based gels, but yield significantly better MS results. Thus, the Bis/ethylene-glycol-diacrylate SDS-PAGE gel system is a promising alternative for MS-based in-gel workflows and might be transferred to other gel-electrophoretic applications.

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.

The metabotropic glutamate receptor 4 (mGluR4) is a G-protein-coupled receptor that mediates inhibition of neurotransmitter release. Here, we used a proteomic approach to identify novel interaction partners of mGluR4 and report that the cytoplasmic C-terminal tail of mGluR4 interacts with microtubule-associated protein 1B (MAP1B). Binding of MAP1B to mGluR4 is inhibited by Ca(2+)/calmodulin, and MAP1B and mGluR4 colocalize at excitatory synapses in cultured hippocampal neurons. Thus, MAP1B might be implicated in the synaptic trafficking and/or regulation of mGluR4.

Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-{methyl[1-(2-naphthoyl)piperidin-4-yl]amino}carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC50 of ∼600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca2+ mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.

Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the αβ heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both α and β subunits. Collectively, our data establish oxidation–ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.

Phosphatidylethanolamines (PEs) are abundant lipid constituents of the cellular membrane. The amino group of PEs exhibits high reactivity with hypochlorous acid that is generated under inflammatory conditions in vivo. The analysis of the resulting PE mono- and dichloramines is of significant interest since these species represent important mediators of lipid peroxidation. We have shown in a previous communication that mass spectrometric detection of PE chloramines is only possible with ESI MS, whereas MALDI-TOF MS fails to detect these products if standard matrices are used. In this work we demonstrate that the detection of PE chloramines is also possible by MALDI-TOF MS if 4-chloro-alpha-cyanocinnamic acid is used as matrix. The underlying processes leading to ionization of these species will be discussed in detail. Both, experimental and theoretical studies taking into account possible intramolecular rearrangements were performed to clarify these aspects.

Treatment of RNA oligonucleotides with strong acids at pH 1-2 rapidly leads to hydrolysis of the phosphodiester bonds at the 5'-position of ribose. Analysis of the resulting degradation products by MALDI coupled to an Orbitrap high resolution mass spectrometer shows almost complete mass ladders from both sides of the nucleotides without interfering fragments from base losses or internal fragments. From the mass differences between adjacent peaks of a mass ladder, the sequence can be determined. Low cleavage efficiency at the termini leads to 2mers and 3mers which can be identified by MS/MS. In this way the complete sequences of different siRNA 21mer single and double strands could be verified. This simple and fast method can be applied for controlling sequences of synthetic oligomers, as well as for de-novo sequencing. Moreover, the method is applicable for localization and identification of RNA modifications as demonstrated using the examples of an oligonucleotide with phosphorothioate backbone and of one containing 2'-methoxy-ribose modifications.

The presynaptic proteome controls neurotransmitter release and the short and long term structural and functional dynamics of the nerve terminal. Using a monoclonal antibody against synaptic vesicle protein 2 we immunopurified a presynaptic compartment containing the active zone with synaptic vesicles docked to the presynaptic plasma membrane as well as elements of the presynaptic cytomatrix. Individual protein bands separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were subjected to nanoscale-liquid chromatography electrospray ionization-tandem mass spectrometry. Combining this method with 2-dimensional benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight and immunodetection we identified 240 proteins comprising synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the pre-synapse. Four maxi-channels, three isoforms of voltage-dependent anion channels and the tweety homolog 1 were co-isolated with the docked synaptic vesicles. As revealed by in situ hybridization, tweety homolog 1 reveals a distinct expression pattern in the rodent brain. Our results add novel information to the proteome of the presynaptic active zone and suggest that in particular proteins potentially involved in the short and long term structural modulation of the mature presynaptic compartment deserve further detailed analysis.

The numerous genes and proteins encoded in the borrelial genome have been shown to undergo differential expression in response to environmental cues. To gain a better understanding of possible interactions between antimicrobial agents and Borrelia , we investigated here the effects of increasing concentrations of penicillin G and doxycycline on the protein expression of the Borrelia burgdorferi s.s. isolate LW2 after 24 and 48 h of incubation. For 14 protein spots in Borrelia exposed to penicillin G at 0.25 and 0.5 μg/ml and for 5 protein spots in Borrelia exposed to doxycycline at 0.5 and 1 μg/ml, differences in spot intensity were identified by use of high resolution two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). At concentrations of both antimicrobial agents around the median minimal inhibitory concentration (0.5 μg/ml), all but one of the detected spots showed a considerable down-regulation as revealed by a ⩾50% decrease of spot intensity in comparison to untreated controls. Most of the spots identified thus far belong to proteins that are encoded by genes localized on the borrelial chromosome and are known to be involved in the different pathways of bacterial cell metabolism. Interestingly, one spot, identified as triosephosphate isomerase, was clearly up-regulated in the presence of doxycycline. Our data provide for the first time scientific evidence that B. burgdorferi s.l., although it possesses a small genome and extremely limited biosynthetic capabilities, shows a variable but distinct physiological response to exposure with penicillin G and doxycycline.

Matrix-assisted laser desorption ionization (MALDI) has become an enabling technology for the fields of protein mass spectrometry (MS) and proteomics. Despite its widespread use, for example, in protein identification via peptide mass fingerprinting, a comprehensive model for the generation of free gas-phase ions has not yet been developed. All matrices in use today, such as α-cyano-4-hydroxycinnamic acid (CHCA), have been found empirically and stem from the early days of MALDI. By systematic and targeted variation of the functional groups of the α-cyanocinnamic acid core unit, 4-chloro-α-cyanocinnamic acid (Cl-CCA) was selected and synthesized, and it exhibited outstanding matrix properties. Key features are a substantial increase in sensitivity and a considerably enhanced peptide recovery in proteomic analyses because of a much more uniform response to peptides of different basicity. Using Cl-CCA as a matrix for a 1 fmol bovine serum albumin (BSA) in-solution digest, the sequence coverage is raised to 48%, compared with 4% for CHCA. For a gel band containing 25 fmol of BSA, unambiguous protein identification becomes possible with Cl-CCA. These findings also imply ion formation via a chemical ionization mechanism with proton transfer from a reactive protonated matrix species to the peptide analytes. The considerable increase in performance promises to have a strong impact on future analytical applications of MALDI, because current sensitivity limits are overcome and more comprehensive analyses come into reach.

Study of the bacterial membrane proteome is a field of growing interest in the research of nutrient transport and processing. Pseudomonas sp. strain phDV1, a Gram-negative bacterium selected for its ability to degrade aromatic compounds, was monitored under different growth substrate conditions, using lysogeny broth medium (LB), glucose, and phenol as sole carbon source. The aim of this study was to characterize the membrane subproteomes of the Pseudomonas strain by proteomic means to assess the protein composition of this subcellular compartments, which appears fundamental for the biodegradation of aromatic compounds. A total number of 129 different proteins have been identified by MALDI-TOF/TOF, 19 of which are membrane proteins that belong to the inner membrane and 10 that belong to the outer membrane. Two membrane proteins were only expressed in the presence of the aromatic substrate. We identified a membrane protein involved in aromatic hydrocarbon degradation as well as a probable porin which may, in fact, function as an aromatic compound-specific porin. Although the presence of different transporters have been reported for different aromatic compounds such as toluene and benzoic acid, to our knowledge, these are the first phenol-inducible membrane transporters identified.

While MALDI-MS is widely accepted for quality control of synthetic oligonucleotides, this method has been regarded as not applicable for a control of the purity and correct annealing of double strands. The results presented here show that the double-strand intensities measured by MALDI-MS maintain and reflect the solution conditions. Using a single-stranded RNA as internal standard, the double-strand intensity can be determined by measuring the intensity ratio of the single strands to the standard under "native" conditions and after denaturation with formic acid. For siRNAs with fully matched 20-21 base pairs, relative intensities of the double strands are between 94 and 97.2%. The stability determined by MALDI-MS for different RNA duplexes correlates well with calculated T m values and the content of G-C pairs. Furthermore, the quantification method enables one to determine an excess of one single strand and the contribution of duplex formation by truncated strands. The results show that MALDI-MS is a fast and reliable method for quality control of synthetic siRNA.

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30–37 °C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

Boswellia serrata resin dry extract is among the few herbal remedies designated with an orphan drug status for the treatment of peritumoral brain edema. In addition, boswellic acids (BAs), the main active ingredients of B. serrata extracts, have potent anti-inflammatory properties, and may represent promising agents for the treatment of inflammatory diseases. Pharmacokinetic studies, however, revealed poor bioavailability, especially of 11-keto-beta-boswellic acid (KBA) and 3-acetyl-11-keto-beta-boswellic acid (AKBA), the most potent BAs. To address the question of whether BAs are extensively metabolized, we determined the metabolic stability of KBA and AKBA in vitro, investigated the in vitro metabolism of BAs, and compared the metabolic profiles of KBA and AKBA with those obtained in rats in vivo. In rat liver microsomes and hepatocytes as well as in human liver microsomes, we found that KBA but not AKBA undergoes extensive phase I metabolism. Oxidation to hydroxylated metabolites is the principal metabolic route. In vitro, KBA yielded metabolic profiles similar to those obtained in vivo in rat plasma and liver, whereas no metabolites of AKBA could be identified in vivo. Furthermore, AKBA is not deacetylated to KBA. This study indicates that the efficacy of B. serrata extract may be enhanced by increasing the bioavailability of AKBA.

Alkene 3, a TBS- and Cbz-protected derivative of vinylglycinol, safely available from methionine, undergoes efficient Heck coupling reactions in high yields. The resulting products can be converted into enantiopure α-amino acids with aromatic and heteroaromatic side-chains. Compound 6a, a brightly fluorescent Fmoc-protected pyrene amino acid is of special interest. Tripeptide 8a, composed of this building block and two arginines, binds to TAR, an RNA element regulating transcriptional efficiency in HIV, with a Kd value of 50 nM. This compound also inhibits a coupled cell-free transcription-translation assay (IC50 = 40 μM) and shows pronounced antibacterial activity against B. subtilis. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Aims: Evaluation of the activity profiles of standard prokaryotic translation inhibitors with different physicochemical properties under whole-cell and cellfree conditions. Methods and Results: The minimal inhibitory concentration values (cellfree ⁄whole-cell l gm l )1 ) for three aminoglycosides (neomycin, 0AE01 ⁄6AE92; paromomycin, 0AE7 ⁄1AE96; streptomycin 1AE45 ⁄1AE57), three macrolides (erythromycin, 1AE53 ⁄56AE9; josamycin, 1AE61 ⁄87AE7; oleandomycin, 5AE12 ⁄565AE9), chloramphenicol (11AE9 ⁄3AE04), and two tetracyclines (tetracycline hydrochloride, not determined ⁄0AE63; minocycline hydrochloride, 2AE53 ⁄1AE09), towards Escherichia coli A19 cells were determined with a microtitre plate-based broth dilution method and compared with values determined in a coupled transcription ⁄translation system based on a S30 extract of the same E. coli strain (cellfree) for the production of the green fluorescent protein. Conclusions: The analysed prokaryotic translation inhibitors showed substance-specific activity profiles under cell-free vs whole-cell conditions that are explainable by the physicochemical properties of the molecules. Significance and Impact of the Study: This study shows the advantages and limits of cell- free transcription ⁄translation (CFTT) experiments for the discovery of novel antimicrobials. The main advantage is the direct access of the target structures (ribosomes) for the inhibitors, and our results provide an estimation of the concentration necessary to detect new agents. The main limitations are that the inhibitory properties of different agents in CFTT experiments do not necessarily reflect their growth inhibition activity in cell cultures.

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.

Das Wachstum der Proteomics hat in den letzten Jahren die Entwicklung massenspektrometrischer Instrumente und Methoden angetrieben. Der Weg vom Massenspektrum zu biologischer Information ist aber nach wie vor weit und Grundlagenforschung bleibt unverzichtbar – ein Uberblick uber aktuelle Trends in der Massenspektrometrie.

Purification methods for proteomics samples are of crucial concern for improving the quality of the sample delivered to the mass spectrometer. They constitute the link between the mass spectrometer and protein processing and peptide isolation steps that usually require solvents, buffers, or detergents completely incompatible with MS-analysis conditions. This work describes three new clean-up procedures using synthetic membranes and polymer media and compares them with standard procedures. The efficiency of each of the purification procedures was studied via application to four standards and two membrane proteins. This work highlights the importance of versatility in sample preparation, especially for MS-based proteomic investigations.

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.

Today, toxicoproteomics still relies mainly on 2-DE followed by MS for detection and identification of proteins, which might characterize a certain state of disease, indicate toxicity or even predict carcinogenicity. We utilized the classical 2-DE/MS approach for the evaluation of early protein biomarkers which are predictive for chemically induced hepatocarcinogenesis in rats. We were able to identify statistically significantly deregulated proteins in N-nitrosomorpholine exposed rat liver tissue. Based on literature data, biological relevance in the early molecular process of hepatocarcinogenicity could be suggested for most of these potential biomarkers. However, in order to ensure reliable results and to create the prerequisites necessary for integration in routine toxicology studies in the future, these protein expression patterns need to be prevalidated using independent technology platforms. In the current study, we evaluated the usefulness of iTRAQ reagent technology (Applied Biosystems, Framingham, USA), a recently introduced MS-based protein quantitation method, for verification of the 2-DE/MS biomarkers. In summary, the regulation of 26 2-DE/MS derived protein biomarkers could be verified. Proteins like HSP 90-beta, annexin A5, ketohexokinase, N-hydroxyarylamine sulfotransferase, ornithine aminotransferase, and adenosine kinase showed highly comparable fold changes using both proteomic quantitation strategies. In addition, iTRAQ analysis delivered further potential biomarkers with biological relevance to the processes of hepatocarcinogenicity: e.g. placental form of glutathione S-transferase (GST-P), carbonic anhydrase, and aflatoxin B1 aldehyde reductase. Our results show both the usefulness of iTRAQ reagent technology for biomarker prevalidation as well as for identification of further potential marker proteins, which are indicative for liver hepatocarcinogenicity.

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.

The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network., Author Summary More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.

Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRbeta subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5-40 amino acids, are subject to alternative splicing (C1-C7, C4'-C6'). Since one of the cassettes, C5', has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRbeta loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6', already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5' was coisolated mainly from liver. Notably C5'-containing gephyrin bound to the GlyRbeta loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.

The F1F0 ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F-1 and subunits a and c of F-0. Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F-1 and F-0. In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the 6 subunit. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

Borrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H-like protein-1 (FHL-1) allows spirochetes to resist complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP-2. BbCRASP-2 is distinct from the four previously identified factor H/FHL-1-binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP-2, cspZ, is located on the linear plasmid lp28-3. BbCRASP-2 is highly divergent from the factor H/FHL-1-binding protein BbCRASP-1 and from members of the factor H-binding Erp (OspE/F-related) protein family. Peptide mapping analysis revealed that the factor H/FHL-1 binding site is discontinuous and it was found that C-terminal truncations abrogate factor H and FHL-1 binding. The predominant BbCRASP-2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL-1 bound to BbCRASP-2 maintain cofactor activity for factor I-mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP-2 in serum-sensitive B. burgdorferi mutant B313 increased resistance to complement-mediated lysis. The characterization of BbCRASP-2 now provides a complete picture of the three diverse complement regulator-binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.

The incorporation of analytes into matrix crystals and even more so its mechanistic aspects as a prerequisite for a successful MALDI-MS has been discussed controversially in the literature. Solventless sample preparation techniques can shed new light on this question. In order to investigate some crucial aspects of these preparation techniques, lyophylized peptides and proteins were ground or milled with the powder of two different matrices, 2,5-DHB as incorporating matrix and 2,6-DHB for which protein incorporation was definitely excluded in a prior study, and pressed into pellets. The dependence of the quality of the UV-MALDI-spectra on the mass (up to 12,360 Da) and the milling time in a ball mill is reported. For mellitin different initial axial ion velocities were found, when desorbed from 2,5-DHB-pellets as prepared and after wetting and re-drying. Velocities of 150 and 580 m s−1 for dry and wetted pellets are taken as representative for hard desorption from a surface and soft desorption of matrix-incorporated analytes, respectively. Proteins labeled with either fluorescein isothiocyanate (FITC) or Texas Red (TR) were nano-electrosprayed onto a bed of ferulic acid in a ‘dry’ or ‘wet’ mode. All ‘dry’ deposits exhibit strong fluorescence but do not yield MALDI-ion signals. All ‘wet’ deposits yield MALDI-signals of the proteins; the fluorescence of FITC is quenched in ‘wet’ deposits because of the low matrix pH.

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MSn analysis performed on a hybrid linear ion trap–orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants. Copyright © 2006 John Wiley & Sons, Ltd.

Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides.

Pathological pain associated either with peripheral tissue damage and inflammation (inflammatory pain) or peripheral nerve injury (neuropathic pain) is characterized by persistent pain hypersensitivity. This hypersensitivity is believed to be mediated by sensitization of nociceptors and spinal dorsal horn neurons leading to hyperalgesia and allodynia. Changes of protein expression and/or phosphorylation are known to contribute to the development of this hyperexcitability of the nociceptive system. In the present study we analyzed protein patterns in the spinal cord following paw inflammation or sciatic nerve injury using two-dimensional (2D) gel electrophoresis combined with MALDI-TOF mass spectrometry. 2D-PAGE revealed nine and five regulated proteins following paw inflammation and sciatic nerve damage, respectively. These regulated proteins had not been identified previously with other methods. There was no overlap of regulated proteins between models except for the small heat shock protein alpha-crystallin, which was decreased in both models. In conclusion, this study illustrates that employment of the proteomic 2D-PAGE approach allows for identification of novel regulated proteins that may be involved in the central sensitization and possibly manifestation of chronic pain.

Our current approach focused on the identification of potential early protein biomarker signatures which are indicative of the carcinogenic processes in rats exposed to 20 mg/kg of the liver carcinogen N-nitrosomorpholine (NNM). Treated liver was investigated at different timepoints. Therefore, proteins were separated by two-dimensional gel electrophoresis as a first step prior to identification of differentially expressed proteins by mass spectrometry. Proteomic analysis of liver samples after one day of exposure revealed significant upregulation of proteins involved in response to cellular stress induced by NNM (superoxide dismutase, heat shock protein 60, peroxiredoxin). Eighteen weeks after withdrawal of NNM, we were able to identify cancer-related proteins in rat liver bearing malignant, transformed cells (caspase-8 precursor, vimentin, Rho GDP dissociation inhibitor). Some of these proteins were already deregulated after three weeks of exposure indicating their potential usefulness as early predictive biomarkers for liver carcinogenicity (annexin A5, fructose-1,6-bisphosphatase). As regulatory toxicology approaches usually include the investigation of carcinogenicity in two-years studies in rodents, especially the detection of early protein biomarker signatures which precede the appearance of neoplasia, demonstrates the high potential of proteomics approaches to substantially reduce the time and costs of carcinogenicity testing.