We report a mass spectrometry-based comparative “bottom up” proteomics approach that combines d0/d4-succinic anhydride labeling with commercially available hydrazine (Hz)-functionalized beads (Affi-gel Hz beads) for detection, identification and relative quantification of site-specific oxylipid modifications in biological matrices. We evaluated and applied this robust and simple method for the quantitative analysis of oxylipid protein conjugates in cardiac mitochondrial proteome samples isolated from 3- and 24-month-old rat hearts. The use of d0/d4-succinic anhydride labeling, Hz-bead based affinity enrichment, nanoLC fractionation and MALDI-ToF/ToF tandem mass spectrometry yielded relative quantification of oxylipid conjugates with residue-specific modification information. Conjugation of acrolein (ACR), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-noneal (ONE) to cysteine, histidine and lysine residues were identified. HHE conjugates were the predominant subset of Michael-type adducts detected in this study. The HHE conjugates showed higher levels in mitochondrial preparations from young heart congruent with previous findings by others that the n-3/n-6 PUFA ratio is higher in young heart mitochondrial membranes. Although this study focuses on protein adducts of reactive oxylipids the method might be equally applicable to protein carbonyl modifications caused by metal catalyzed oxidation reactions.
Protein carbonyls; oxidative stress; lipid peroxidation products; mitochondria; mass spectrometry; heart
The establishment of extremely powerful proteomics platforms able to map thousands of modification sites, e.g. phosphorylations or acetylations, over entire proteomes calls for equally powerful software tools to effectively extract useful and reliable information from such complex datasets. We present a new quantitative PTM analysis platform aimed at processing iTRAQ or Tandem Mass Tags (TMT) labeled peptides. It covers a broad range of needs associated with proper PTM ratio analysis such as PTM localization validation, robust ratio computation and statistical assessment, and navigable user report generation. IsobarPTM is made available as an R Bioconductor package and it can be run from the command line by non R specialists.
“IsobarPTM is a new software tool facilitating the quantitative analysis of protein modification regulation streamlining important issues related to PTM localization and statistical modeling. Users are provided with a navigable spreadsheet report, which also annotate already public modification sites.” This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations.
•IsobarPTM is a new open source software tool to analyze PTM regulation.•Quantitative iTRAQ and TMT data are supported.•Whole set of functionalities from PTM size localization to statistical modeling•Navigable user reports are generated automatically.•Available as R Bioconductor package
Bioinformatics; Computational proteomics; Quantitative proteomics; iTRAQ; TMT; Statistics
Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS could revolutionize neuronal network and biomarker discovery studies.
Imaging mass spectrometry; MALDI; neurons; neuroendocrine tissues; sampling; neuropeptides
Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies—secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI MS)—are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enable new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.
secondary ion mass spectrometry; matrix assisted laser desorption/ionization; subcellular profiling; elemental imaging; mass cytometry; tissue imaging
Many microbes can be cultured as single-species communities. Often, these colonies are controlled and maintained via the secretion of metabolites. Such metabolites have been an invaluable resource for the discovery of therapeutics (e.g. penicillin, taxol, rapamycin, epothilone). In this article, written for a special issue on imaging mass spectrometry, we show that MALDI-imaging mass spectrometry can be adapted to observe, in a spatial manner, the metabolic exchange patterns of a diverse array of microbes, including thermophilic and mesophilic fungi, cyanobacteria, marine and terrestrial actinobacteria, and pathogenic bacteria. Dependent on media conditions, on average and based on manual analysis, we observed 11.3 molecules associated with each microbial IMS experiment, which was split nearly 50:50 between secreted and colony-associated molecules. The spatial distributions of these metabolic exchange factors are related to the biological and ecological functions of the organisms. This work establishes that MALDI-based IMS can be used as a general tool to study a diverse array of microbes. Furthermore the article forwards the notion of the IMS platform as a window to discover previously unreported molecules by monitoring the metabolic exchange patterns of organisms when grown on agar substrates.
The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology.
Phosphorylation; Mitochondria; Cardiovascular Disease; Protein Quantitation; MRM
Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible.
Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far.
This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
•Quantitative cross-linking using isotope labelling is technically feasible•Commercial cross-linker BS3-d0/4 is suitable for quantitative cross-linking•XiQ, an open source script to extract quantitative data from raw files•Cross-link reference data set to test identification and quantitation software•Isotope labelling reduced the number of identified cross-links
Quantitation; Cross-linking; Structural biology; Protein dynamics; Mass spectrometry; Proteomics
Mass spectrometry, in the past five years, has increased in speed, accuracy and use. With the ability of the mass spectrometers to identify increasing numbers of proteins the identification of undesirable peptides (those not from the protein sample) has also increased. Most undesirable contaminants originate in the laboratory and come from either the user (e.g. keratin from hair and skin), or from reagents (e.g. trypsin), that are required to prepare samples for analysis. We found that a significant amount of MS instrument time was spent sequencing peptides from abundant contaminant proteins. While completely eliminating non-specific protein contamination is not feasible, it is possible to reduce the sequencing of these contaminants. For example, exclusion lists can provide a list of masses that can be used to instruct the mass spectrometer to ‘ignore’ the undesired contaminant peptides in the list. We empirically generated be-spoke exclusion lists for several model organisms (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae and Xenopus laevis), utilising information from over 500 mass spectrometry runs and cumulative analysis of these data. Here we show that by employing these empirically generated lists, it was possible to reduce the time spent analysing contaminating peptides in a given sample thereby facilitating more efficient data acquisition and analysis.
Given the current efficacy of the Mass Spectrometry instrumentation, the utilisation of data from ~500 mass spec runs to generate be-spoke exclusion lists and optimise data acquisition is the significance of this manuscript.
This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
•We looked at the contribution of ‘contaminating’ peptides to MS acquisition time.•We found that 30–50% of time was wasted on contaminant peptide sequencing.•We tested whether the application of an exclusion list would solve this problem.•Exclusion lists generated for specific species from > 500 mass spectrometry runs.•Exclusion lists enabled more efficient analysis with coverage and isoform data.
HPLC-MS/MS, high pressure liquid chromatography-tandem mass spectrometry; ACN, acetonitrile; PEG, polyethylene glycol; BSA, bovine serum albumin; PTM, post translation modification; MS, mass spectrometry; MRM, multiple reaction monitoring; SRM, selective reaction monitoring; CID, collision induced dissociation; m/z, mass-to-charge ratio; SILAC, stable isotope labelling by amino acids in cell culture; IP, immuno-precipitation; Contamination; Data analysis; Exclusion list; MS optimisation
Ticks are mostly obligatory blood feeding ectoparasites that have an impact on human and animal health. In addition to direct damage due to feeding, some tick species serve as the vectors for the causative agents of several diseases, such as the spirochetes of the genus Borrelia causing Lyme disease, the virus of tick-borne encephalitis, various Rickettsial pathogens or even protozoan parasites like Babesia spp. Hard ticks are unique among bloodfeeders because of their prolonged feeding period that may last up to two weeks. During such a long period of blood uptake, the host develops a wide range of mechanisms to prevent blood loss. The arthropod ectoparasite, in turn, secretes saliva in the sites of bite that assists blood feeding. Indeed, tick saliva represents a rich source of proteins with potent pharmacologic action that target different mechanisms of coagulation, platelet aggregation and vasoconstriction. Tick adaptation to their vertebrate hosts led to the inclusion of a powerful protein armamentarium in their salivary secretion that has been investigated by high throughput methods. The resulting knowledge can be exploited for the isolation of novel antihemostatic agents. Here we review the tick salivary antihemostatics and their characterized functions at the molecular and cellular levels.
tick; salivary gland; hemostasis; coagulation; platelet aggregation; serine proteases; thrombin; kunitz-domain protein; disintegrin; serpin
Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (<1% of total protein phosphorylation), only a few tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca2+ homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states.
Tyrosine phosphorylation; Rat muscle; Tyrosine immunoprecipitation; HPLC-ESI-MS/MS
A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-δ). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.
PMID: 19393779 CAMSID: cams3168
Apoptosis; Camptothecin; Lysosome; iTRAQ reagent; LC-ESI-MS/MS; Confocal Microscopy
Cardiovascular diseases (CVDs) are the major source of global morbidity and death and more people die annually from CVDs than from any other cause. These diseases can occur quickly, as seen in acute myocardial infarction (AMI), or progress slowly over years as with chronic heart failure. Advances in mass spectrometry detection and analysis, together with improved isolation and enrichment techniques allowing for the separation of organelles and membrane proteins, now allow for the in-depth analysis of the cardiac proteome. Here we outline current insights that have been provided through cardiovascular proteomics, and discuss studies that have developed innovative technologies which permit the examination of the protein complement in specific organelles including exosomes and secreted proteins. We highlight these foundational studies and illustrate how they are providing the technologies and tools which are now being applied to further study cardiovascular disease; provide new diagnostic markers and potentially new methods of cardiac patient management with identification of novel drug targets.
PMID: 23153792 CAMSID: cams2799
Accurate and reliable quantitative proteomics in cell culture has been considerably facilitated by the introduction of the stable isotope labeling by amino acids in cell culture (SILAC), combined with high resolution mass spectrometry. There are however several major sources of quantification errors that commonly occur with SILAC techniques, i.e. incomplete incorporation of isotopic amino acids, arginine-to-proline conversion, and experimental errors in final sample mixing. Dataset normalization is a widely adopted solution to such errors, however this may not completely prevent introducing incorrect expression ratios. Here we demonstrate that a label-swap replication of SILAC experiments was able to effectively correct experimental errors by averaging ratios measured in individual replicates using quantitative proteomics and phosphoproteomics of ligand treatment of neural cell cultures. Furthermore, this strategy was successfully applied to a SILAC triplet experiment, which presents a much more complicated experimental matrix, affected by both incomplete labeling and arginine-to-proline conversion. Based on our results, we suggest that SILAC experiments should be designed to incorporate label-swap replications for enhanced reliability in expression ratios.
SILAC; incomplete isotope labeling; arginine-to-proline conversion; label-swap replication; receptor
Peptide quantification using MS often relies on the comparison of peptide signal intensities between different samples, which is based on the assumption that observed signal intensity has a linear relationship to peptide abundance. A typical proteomics experiment is subject to multiple sources of variance, so we focussed here on properties affecting peptide linearity under simple, well-defined conditions. Peptides from a standard protein digest were analysed by multiple reaction monitoring (MRM) MS to determine peptide linearity over a range of concentrations. We show that many peptides do not display a linear relationship between signal intensity and amount under standard conditions. Increasing the organic content of the sample solvent increased peptide linearity by increasing the accuracy and precision of quantification, which suggests that peptide non-linearity is due to concentration-dependent surface adsorption. Using multiple peptides at various dilutions, we show that peptide non-linearity is related to observed retention time and predicted hydrophobicity. Whereas the effect of adsorption on peptide storage has been investigated previously, here we demonstrate the deleterious effect of peptide adsorption on the quantification of fresh samples, highlight aspects of sample preparation that can minimise the effect, and suggest bioinformatic approaches to enhance the selection of peptides for quantification.
Accurate quantification is central to many aspects of science, especially those examining dynamic processes or comparing molecular stoichiometries. In biological research, the quantification of proteins is an important yet challenging objective. Large-scale quantification of proteins using MS often depends on the comparison of peptide intensities with only a single-level calibrant (as in stable isotope labelling and absolute quantification approaches) or no calibrants at all (as in label-free approaches). For these approaches to be reliable, it is essential that the relationship between signal intensity and concentration is linear, without a significant intercept. Here, we show that peptide adsorption can severely affect this relationship, even under controlled conditions, and we demonstrate simple methodologies that can be used to moderate and predict this effect. These findings thus enable the quantification of proteins with increased robustness and reliability.
•First description of effect of peptide adsorption on linearity and quantification•Demonstration that peptide adsorption affects quantification of fresh samples•Addition of acetonitrile improves peptide solubility and reliability of quantification
MRM, multiple reaction monitoring; v/v, volume per volume; Abundance; Linearity; MS; Peptide; Quantification
Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry were employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu4+-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.
PC12 cells; 2-D gel electrophoresis; proteomics; plutonium-binding proteins; IMAC; anti-apoptotic; GO process
Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, Thr22, Thr453, Ser478, Thr671, Ser678, and Ser680) and quantified 16 of them under basal and insulin stimulated conditions in hamster ovary cells overexpressing the insulin receptor (CHO/IR), an insulin sensitive cell model. Insulin stimulated the phosphorylation of PPP1R12A significantly at Ser477, Ser478, Ser507, Ser668, and Ser695, while simultaneously suppressing the phosphorylation of PPP1R12A at Ser509 (more than 2-fold increase or decrease compared to basal). Our data demonstrate that PPP1R12A undergoes insulin stimulated/suppressed phosphorylation, suggesting that PPP1R12A phosphorylation may play a role in insulin signal transduction. The novel PPP1R12A phosphorylation sites as well as the new insulin-responsive phosphorylation sites of PPP1R12A in CHO/IR cells provides targets for investigation of the regulation of PPP1R12A and the PPP1R12A-PP1cδ complex in insulin action and other signaling pathways in other cell models, animal models, and humans.
PPP1R12A; phosphorylation; HPLC-ESI-MS/MS; quantification
Recent emergences of glycobiology, glycotechnology and glycomics have been clarifying enormous roles of carbohydrates in biological recognition systems. For example, cell surface carbohydrates existing as glycoconjugates (glycolipids, glycoproteins and proteoglycans) play crucial roles in cell-cell communication, cell proliferation and differentiation, tumor metastasis, inflammatory response or viral infection. In particular, sialic acids (SAs) existing as terminal residues in carbohydrate chains on cell surface are involved in signal recognition and adhesion to ligands, antibodies, enzymes and microbes. In addition, plasma free SAs and sialoglycans have shown great potential for disease biomarker discovery. Therefore, development of efficient analytical methods for structural and functional studies of SAs and sialylglycans are very important and highly demanded. The problems of SAs and sialylglycans analysis are vanishingly small sample amount, complicated and unstable structures, and complex mixtures. Nevertheless, in the past decade, mass spectrometry in combination with chemical derivatization and modern separation methodologies has become a powerful and versatile technique for structural analysis of SAs and sialylglycans. This review summarizes these recent advances in glycomic studies on SAs and sialylglycans. Specially, derivatization and capturing of SAs and sialylglycans combined with mass spectrometry analysis are highlighted.
Insulin resistance is a major pathophysiologic abnormality that characterizes metabolic syndrome and type 2 diabetes. A well characterized ethanolic extract of Artemisia dracunculus L., termed PMI 5011, has been shown to improve insulin action in vitro and in vivo, but the cellular mechanisms remain elusive. Using differential proteomics, we have studied mechanisms by which PMI 5011 enhances insulin action in primary human skeletal muscle culture obtained by biopsy from obese, insulin-resistant individuals. Using iTRAQ™ labeling and LC-MS/MS, we have identified over 200 differentially regulated proteins due to treatment with PMI 5011 and insulin stimulation. Bioinformatics analyses determined that several metabolic pathways related to glycolysis, glucose transport and cell signaling were highly represented and differentially regulated in the presence of PMI 5011 indicating that this extract affects several pathways modulating carbohydrate metabolism, including translocation of GLUT4 to the plasma membrane. These findings provide a molecular mechanism by which a botanical extract improves insulin stimulated glucose uptake, transport and metabolism at the cellular level resulting in enhanced whole body insulin sensitivity.
botanicals; insulin resistance; iTRAQ; proteomics; skeletal muscle; glucose transporter 4
Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers’ regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.
Carica papaya; Label-free quantitative proteomics; Latex; Mass spectrometry; Plant proteomics
Nucleoid-associated proteins (NAPs) are small, highly abundant transcriptional regulators with low sequence specificity which are involved in multiple DNA-related processes including gene expression, DNA protection, recombination/repair and nucleoid structuring. Through these functions they are able to regulate important phenotypic properties including virulence, secondary metabolism and stress resistance. However the set of NAPs known within the Actinobacteria is small and incomplete. The missing proteins are likely to be key regulators of virulence in pathogens such as Mycobacterium tuberculosis and also of development and secondary metabolism in industrially-important species such as Streptomyces. Here, we use label-free LC–MS/MS to systematically search for novel NAPs in isolated nucleoids of the model actinomycete Streptomyces coelicolor. Based on the criteria of high abundance (emPAI score) and predicted DNA-binding ability (DNAbinder score) we identified a set of 24 proteins with a high predicted likelihood of being NAPs. The approach was deemed successful as the set included known major NAPs HupA, HupS, sIHF and Lsr2 as well as the global transcriptional regulators BldD and CRP and the pleiotropic response regulator AfsQ1. It also included a number of proteins whose functions are not yet known from recognisable classes of transcription factor (SCO2140, SCO4493, SCO1839, SCO1210, SCO5405, SCO4229, SCO3198) or from uncharacterised protein families (SCO5783, SCO5592, SCO3793, SCO6482) which comprise a valuable set of candidates for further study.
In this paper we establish a robust protocol for preparing S. coelicolor nucleoids for mass spectrometric analysis and develop a workflow for identifying novel nucleoid-associated proteins (NAPs) by combining LC–MS/MS with a bioinformatical analysis. The nucleoid-associated proteins of many species are known to be key regulators of virulence, stress tolerance and global patterns of gene expression. Identifying the “missing” nucleoid proteins of S. coelicolor is likely to have important implications for manipulating the production of secondary metabolites such as antibiotics. Candidate NAPs were identified. Several of these are highly conserved in clinically important species such as Mycobacterium and in many commercially important species such as Salinispora and Micromonospora which represent a vital source of novel drugs such as antibiotics, antifungals and anticancer agents.
•Streptomyces coelicolor was grown in liquid culture to late vegetative phase.•Whole nucleoids were isolated by sucrose gradient sedimentation.•Proteins attached to the nucleoids were identified by label-free LC–MS/MS.•A list of high-abundance DNA-binding proteins was generated, representing likely NAPs.
GR, Global regulator; NAP, Nucleoid-associated proteins; DNA-binding; LC–MS/MS; Nucleoid; H-NS; IHF; Streptomyces
Pulsed Q collision-induced dissociation (PQD) was developed in part to facilitate detection of low-mass reporter ions using labeling reagents (e.g. iTRAQ) on LTQ platforms. It has generally been recognized that the scan speed and sensitivity of an LTQ are superior than those of an Orbitrap using the higher-energy collisional dissociation (HCD). However, the use of PQD in quantitative proteomics is limited, primarily due to the meager reproducibility of reporter ion ratios. Optimizations of PQD for iTRAQ quantification using LTQ have been reported, but a universally applicable strategy for quantifying the less abundant proteins has not been fully established. Adjustments of the AGC target, µscan, or scan speed offer only incremental improvements in reproducibility. From our experience, however, satisfactory coefficients of variation (CVs) of reporter ion ratios were difficult to achieve using the discovery-based approach. As an alternative, we implemented a target-based approach that obviates data dependency to allow repetitive data acquisitions across chromatographic peaks. Such a strategy generates enough data points for more reliable quantification. Using cAMP treatment in S49 cell lysates and this target-based approach, we were able to validate differentially expressed proteins, which were initially identified as potential candidates using the discovery-based PQD. The target-based strategy also yielded results comparable to those obtained from HCD in an Orbitrap. Our findings should aid LTQ users who desire to explore iTRAQ quantitative proteomics but have limited access to the more costly Orbitrap or other instruments.
Pulsed Q collision-induced dissociation (PQD); linear ion trap; triple quadrupole (QqQ); higher energy collisional dissociation (HCD); iTRAQ (Isobaric Tag for Relative and Absolute Quantification)
Cortactin is an F-actin binding protein that functions as a scaffold to regulate Arp2/3 mediated actin polymerization in lamellipodia and invadopodia formation as well as functioning in cell migration and endocytosis of many different cell types. In light of the fact that regulated actin polymerization is critical for many cellular processes we launched a search for novel cortactin interactions with cellular proteins that might indicate heretofore undescribed biological activities supported by cortactin. Using a modified stable isotope labeling in cell culture (SILAC) approach in HEK293 cells and Flag-tagged cortactin (F-cortactin) as bait, we identified a limited set of cortactin interactions including several proteins which have not previously been identified as cortactin associated proteins. Among these were serine/threonine-protein phosphatase 2A subunit beta (PP2A-beta) and RCC2/TD60, a Rac guanine nucleotide exchange factor (GEF) required for completion of mitosis and cytokinesis. The interaction between cortactin and RCC2/TD60 was verified in cell lysates immunoprecitated with anti- RCC2/TD60 antibody. Furthermore, cortactin was localized by immunofluorescence in the equatorial plane of dividing HeLa cells in the region where RCC2/TD60 has previously been localized thus providing support for a complex containing cortactin and RCC2/TD60 complex that may play a functional role in cells undergoing mitosis.
SILAC; focal adhesion; cortactin; RCC2/TD60
The VLDL transport vesicle (VTV) mediates the transport of nascent VLDL particles from the ER to the Golgi and plays a key role in VLDL-secretion from the liver. The functionality of VTV is controlled by specific proteins; however, full characterization and proteomic profiling of VTV remain to be carried out. Here, we report the first proteomic profile of VTVs. VTVs were purified to their homogeneity and characterized biochemically and morphologically. Thin section transmission electron microscopy suggests that the size of VTV ranges between 100 nm to 120 nm and each vesicle contains only one VLDL particle. Immunoblotting data indicate VTV concentrate apoB100, apoB48 and apoAIV but exclude apoAI. Proteomic analysis based on 2D-gel coupled with MALDI-TOF identified a number of vesicle-related proteins, however, many important VTV proteins could only be identified using LC-MS/MS methodology. Our data strongly indicate that VTVs greatly differ in their proteome with their counterparts of intestinal origin, the PCTVs. For example, VTV contains Sec22b, SVIP, ApoC-I, reticulon 3, cideB, LPCAT3 etc. which are not present in PCTV. The VTV proteome reported here will provide a basic tool to study the mechanisms underlying VLDL biogenesis, maturation, intracellular trafficking and secretion from the liver.
very low-density lipoprotein (VLDL); VLDL transport vesicle (VTV); apolipoprotein B (apoB); endoplasmic reticulum (ER); triacylglycerol (TAG)
N-glycosylation of proteins provides a rich source of information on liver disease progression because majority of serum glycoproteins, with the exception of immunoglobulins, are secreted by the liver. In this report, we present results of an optimized workflow for MALDI-TOF analysis of permethylated N-glycans detached from serum proteins and separated into liver secreted and immunoglobulin fractions. We have compared relative intensities of N-glycans in 23 healthy controls and 23 cirrhosis patients. We were able to detect 82 N-glycans associated primarily with liver secreted glycoproteins, 54 N-glycans in the protein G bound fraction and 52 N-glycans in the fraction bound to protein A. The N-glycan composition of the fractions differed substantially, independent of liver disease. The relative abundance of approximately 53% N-glycans in all fractions was significantly altered in the cirrhotic liver. The removal of immunoglobulins allowed detection of an increase in a series of high mannose and hybrid N-glycans associated with the liver secreted protein fraction.
N-glycosylation; Mass spectrometry; Serum; Liver disease
The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE) – tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p≤0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially were verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.
Baker s yeast; Cancer prevention; Proteomics; Selenium; Pyruvate kinase