Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339

The ruthenium compound KP1019 has demonstrated promising anticancer activity in a pilot clinical trial. This study aims to evaluate the intracellular uptake/binding patterns of KP1019 and its sodium salt KP1339, which is currently in a phase I–IIa study. Although KP1339 tended to be moderately less cytotoxic than KP1019, IC₅₀ values in several cancer cell models revealed significant correlation of the cytotoxicity profiles, suggesting similar targets for the two drugs. Accordingly, both drugs activated apoptosis, indicated by caspase activation via comparable pathways. Drug uptake determined by inductively coupled plasma mass spectrometry (ICP-MS) was completed after 1 h, corresponding to full cytotoxicity as early as after 3 h of drug exposure. Surprisingly, the total cellular drug uptake did not correlate with cytotoxicity. However, distinct differences in intracellular distribution patterns suggested that the major targets for the two ruthenium drugs are cytosolic rather than nuclear. Consequently, drug–protein binding in cytosolic fractions of drug-treated cells was analyzed by native size-exclusion chromatography (SEC) coupled online with ICP-MS. Ruthenium–protein binding of KP1019- and KP1339-treated cells distinctly differed from the platinum binding pattern observed after cisplatin treatment. An adapted SEC-SEC-ICP-MS system identified large protein complexes/aggregates above 700 kDa as initial major binding partners in the cytosol, followed by ruthenium redistribution to the soluble protein weight fraction below 40 kDa. Taken together, our data indicate that KP1019 and KP1339 rapidly enter tumor cells, followed by binding to larger protein complexes/organelles. The different protein binding patterns as compared with those for cisplatin suggest specific protein targets and consequently a unique mode of action for the ruthenium drugs investigated.     

Development of inductively coupled plasma-mass spectrometry-based protease assays

Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr∼Asp-Lys(biotin) and Lu-DTPA-βAla-βAla-βAla-βAla-Gly-Ser-Ala-Tyr∼Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for α-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2 pM could be readily detected, superior to the detection limit of an assay using the α-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.     

Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

A sample preparation strategy for the determination of the Fe-containing enzyme catalase (CAT) by Fe specific monitoring in human erythrocytes has been optimized. For this purpose, the combined use of elemental mass spectrometry (via inductively coupled plasma, ICP-MS), molecular mass spectrometry (via MALDI-TOF) and enzymatic activity measurements has been required. The procedure involved haemoglobin precipitation from cell lysate with a solution of ethanol-chloroform and preconcentration of the supernatant by using a Speed-Vac concentrator. Catalase recoveries of about 88 ± 15% could be measured by monitoring the protein enzymatic activity before and after precipitation. Further fractionation of Fe-containing proteins from the preconcentrated extract was achieved by size exclusion chromatography (Superdex 200) with a mobile phase of ammonium acetate (0.05 M, pH 7.4) coupled to ICP-MS (Fe monitoring) and UV/VIS detection (specific absorption of the heme-group at 408 nm). A second dimensional chromatography of the CAT-positive activity fraction was carried out by anion-exchange chromatography (Mono Q 5/50) using for elution a linear gradient of ammonium acetate (0-0.750 M in 15 min). This second step revealed a single Fe-containing species in the chromatogram and permitted the unambiguous characterization of the CAT in such fractions by MALDI-TOF. Column recoveries were evaluated and were quantitative, in terms of Fe bound to protein and CAT activity.     

Metaproteome analysis to determine the metabolically active part of a thermophilic microbial community producing biogas from agricultural biomass

Complex consortia of microorganisms are responsible for biogas production. A lot of information about the taxonomic structure and enzymatic potential of such communities has been collected by a variety of gene-based approaches, yet little is known about which of all the assumable metabolic pathways are active throughout the process of biogas formation. To tackle this problem, we established a protocol for the metaproteomic analysis of samples taken from biogas reactors fed with agricultural biomass. In contrast to previous studies where an anaerobic digester was fed with synthetic wastewater, the complex matrix in this study required the extraction of proteins with liquid phenol and the application of paper bridge loading for 2-dimensional gel electrophoresis. Proteins were subjected to nanoHPLC (high-performance liquid chromatography) coupled to tandem mass spectrometry for characterization. Several housekeeping proteins as well as methanogenesis-related enzymes were identified by a MASCOT search and de novo sequencing, which proved the feasibility of our approach. The establishment of such an approach is the basis for further metaproteomic studies of biogas-producing communities. In particular, the apparent status of metabolic activities within the communities can be monitored. The knowledge collected from such experiments could lead to further improvements of biogas production.     

Synthesis and immunoregulatory activities of conjugates of a Toll-like receptor 7 inert ligand

In the synthesis and modification of the analogs of an adenine type of Toll-like receptor (TLR) 7 agonists, we found a special compound, 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine (6). It is a synthesized TLR7 inert ligand, which does not respond to TLR7 itself. However, it can be coupled with protein or peptide antigens via propionyloxy functional group to promote their immunogenicity significantly. The compound was covalently coupled to protein and peptide to get the conjugates. The inductivity of cytokine production by the conjugates was 872.4-fold compared with the unconjugated antigens in vitro by mouse splenocyte. These data show that the immunostimulatory activity of inert TLR7 ligand can be endowed, and the activity of antigens can be amplified by conjugation with various proteins and peptides, thus broadening the potential therapeutic application and reducing the risk of TLR7 agonists’ side effects.     

基于电感耦合等离子体质谱的单细胞分析

单细胞分析可以获得细胞在微环境中准确的个体信息,对于研究细胞的信号传导、生理病理和疾病的早期诊断等具有十分重要的意义.近年来,基于电感耦合等离子体质谱(ICP-MS)的单细胞分析方法开始得到越来越多的应用.本文综述了基于ICP-MS的单细胞分析方法及其在免疫分析、疾病诊断、药物筛选、纳米分析等方面的部分应用,并对基于ICP-MS的单细胞分析方法做出总结和展望.     

Hydrophobic Cluster Analysis of G Protein-Coupled Receptors: A Powerful tool to Derive Structural and Functional Information from 2D-Representation of Protein Sequences

Abstract

Current methods for comparative analyses of protein sequences are 1D-alignments of amino acid sequences based on the maximization of amino acid identity (homology) and the prediction of secondary structure elements. This method has a major drawback once the amino acid identity drops below 20–25 %, since maximization of a homology score does not take into account any structural information. A new technique called Hydrophobic Cluster Analysis (HCA) has been developed by Lemesle-Varloot et al. (Biochimie 72, 555–574), 1990). This consists of comparing several sequences simultaneously and combining homology detection with secondary structure analysis.

HCA is primarily based on the detection and comparison of structural segments constituting the hydrophobic core of globular protein domains, with or without transmembrane domains. We have applied HCA to the analysis of different families of G-protein coupled receptors, such as catecholamine receptors as well as peptide hormone receptors. Utilizing HCA the thrombin receptor, a new and as yet unique member of the family of G-protein coupled receptors, can be clearly classified as being closely related to the family of neuropeptide receptors rather than to the catecholamine receptors for which the shape of the hydrophobic clusters and the length of their third cytoplasmic loop are very different. Furthermore, the potential of HCA to predict relationships between new putative and already characterized members of this family of receptors will be presented.     

基于电感耦合等离子体质谱的蛋白质定量技术研究进展

综述了ICP-MS法应用于蛋白质定量技术方面的研究进展.蛋白质定量研究已成为蛋白质组学研究领域的热点,它是解析生物体蛋白质功能的重要途径.基于同位素标记和生物质谱分析技术是蛋白质定量最常用的方法之一,近年来,随着质谱技术的发展,电感耦合等离子体质谱(ICP-MS)技术成为元素测量的重要手段,这使其在蛋白质定量中具一定的应用前景.     

Quantification of apolipoprotein A-I mimetic peptide D-4F in rabbit plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

The apolipoprotein A-I mimetic peptide D-4F is a potential therapeutical agent effective in maintaining cardiovascular health. A bioanalytical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) to quantitate the D-4F amount in rabbit plasma was developed and validated. A compound with a close structure similarity to the D-4F (only one amino acid A–V altered) was used as an internal standard. Both D-4F and the internal standard were extracted by protein precipitation using acetonitrile/0.2% Triton XL 80N. The correlation coefficient of the calibration curve was 0.9991 in the range 20–40,000 ng/mL. This assay can be used for pharmacokinetic studies of the drug. Also, it may be adjusted for the quantification of other members of apolipoprotein A-I mimetic peptide family.     

Metalloprotein separation and analysis by directly coupled size exclusion high-performance liquid chromatography inductively coupled plasma mass spectroscopy

The feasibility of using directly coupled size exclusion high-performance liquid chromatography inductively coupled plasma mass spectroscopy (HPLC/ICP-MS) for the separation and subsequent elemental analysis of metalloproteins in biological samples has been studied. Data, on up to eight elements, was acquired simultaneously and the reconstructed elemental profiles from the chromatographed samples were quantified by flow injection analysis. Absolute and relative detection limits, reproducibility, operational dynamic range, and linearity of response were initially evaluated by analyzing standards of metallothionein protein of known elemental composition for Cd, Zn, and Cu. There was evidence of displacement of Zn from the protein during chromatography and the substitution of Cu sequestered from the mobile phase. Cd associated with the protein was fully recovered during chromatography. Memory effects, due to protein adsorption to the glassware in the torch box, were minimal and there was no degradation of the resolution of the chromatographed peak during extended transport through the HPLC/ICP-MS interface. The versatility of the technique has been demonstrated by the quantitative multi-element analysis of cytosolic metal-binding proteins separated from the polychaete worm Neanthes arenaceodentata. Fidelity of analysis has been demonstrated by two independent procedures: first, by comparing the elemental profiles obtained by directly aspirating the HPLC eluant into the ICP-MS to those obtained by collecting fractions and quantifying the metal content of the proteins in the conventional analytical mode; second, by comparing the stable isotopic profiles for 114Cd obtained by simultaneous ICP-MS analysis with radiometric profiles of 109Cd obtained by counting radioactivity associated with collected fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

A facile fluorescence turn-on biosensor customized for monitoring of protein kinase activity based on carboxylic carbon nanoparticle–peptide complexes

In this study, we present a facile and low-cost approach for detecting protein kinase A (PKA) by assembling a purpose-designed carboxyfluorescein (FAM)-labelled peptide with carboxylic carbon nanoparticles (CNPs). Fluorescence of the FAM-labelled peptide gradually decreases to a low background signal as a result of the electron transfer from CNPs to FAM-labelled peptide via the peptide, which acts as a bridge. The reaction in the sensor in the presence of adenosine 5′-triphosphate and PKA phosphorylates the substrate peptide and disrupts the electrostatic repulsive force between the CNPs and the peptide, therefore altering the spectroscopic signal of the system. The change in fluorescence signal was directly proportional to the PKA concentration in the range 0–1.8 U/ml with a detection limit of 0.04 U/ml. These results suggest that PKA activity can be effectively measured using the developed PKA biosensor. Moreover, the fluorescence biosensor was successfully used in the investigation of PKA in spiked human embryonic kidney (HEK) 293 cells lysates, indicating its potential applications in protein kinase-related biochemical fundamental research.     

Metallomics investigations on potential binding partners of methylmercury in tuna fish muscle tissue using complementary mass spectrometric techniques

In this study, the binding behaviour of methylmercury (MeHg(+)) towards proteins is investigated. Free sulfhydryl groups in cysteine residues are known to be the most likely binding partners, due to the high affinity of mercury to sulphur. However, detailed knowledge about discrete binding sites in living organisms has been so far scarce. A metallomics approach using different methods like size-exclusion chromatography (SEC) and liquid chromatography (LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) as well as complementary mass spectrometric techniques (electrospray ionisation-tandem mass spectrometry, ESI-MS/MS) are combined to sequence and identify possible target proteins or peptides after enzymatic digestion. Potential targets for MeHg(+) in tuna fish muscle tissue are investigated using the certified reference material CRM464 as a model tissue. Different extraction procedures appropriate for the extraction of proteins are evaluated for their efficiency using isotope dilution analysis for the determination of total Hg in the extracts. Due to the high chemical stability of the mercury-sulphur bond, the bioconjugate can be quantitatively extracted with a combination of tris(hydroxymethyl)aminomethane (TRIS) and sodium dodecyl sulphate (SDS). Using different separation techniques such as SEC and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) it can be shown that major binding occurs to a high-molecular weight protein (M(w) > 200 kDa). A potential target protein, skeletal muscle myosin heavy chain, could be identified after tryptic digestion and capillary LC-ESI-MS/MS.     

Single-cell ICP-MS to address the role of trace elements at a cellular level

The heterogeneity properties shown by cells or unicellular organisms have led to the development of analytical methods at the single-cell level. In this sense, considering the importance of trace elements in these biological systems, the inductively coupled plasma mass spectrometer (ICP-MS) configured for analyzing single cell has presented a high potential to assess the evaluation of elements in cells. Moreover, advances in instrumentation, such as coupling laser ablation to the tandem configuration (ICP-MS/MS), or alternative mass analyzers (ICP-SFMS and ICP-TOFMS), brought significant benefits, including sensitivity improvement, high-resolution imaging, and the cell fingerprint. From this perspective, the single-cell ICP-MS has been widely reported in studies involving many fields, from oncology to environmental research. Hence, it has contributed to finding important results, such as elucidating nanoparticle toxicity at the cellular level and vaccine development. Therefore, in this review, the theory of single-cell ICP-MS analysis is explored, and the applications in this field are pointed out. In addition, the instrumentation advances for single-cell ICP-MS are addressed.     

Versatile convergent synthesis of a three peptide loop containing protein mimic of whooping cough pertactin by successive Cu(I)‐catalyzed azide alkyne cycloaddition on an orthogonal alkyne functionalized TAC‐scaffold

Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein–protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)‐catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

Identification and relative quantitation of an orphan G-protein coupled receptor SREB2 (GPR85) protein in tissue using a linear ion trap mass spectrometer

SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2-overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate.     

TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs

Platinating agents are commonly prescribed anticancer drugs damaging DNA. Induced lesions are recognized by a wide range of proteins. These are involved in cellular mechanisms such as DNA repair, mediation of cytotoxicity or chromatin remodeling. They therefore constitute crucial actors to understand pharmacology of these drugs. To expand our knowledge about this subproteome, we developed a ligand fishing trap coupled to high throughput proteomic tools. This trap is made of damaged plasmids attached to magnetic beads, and was exposed to cell nuclear extracts. Retained proteins were identified by nanoHPLC coupled to tandem mass spectrometry. This approach allowed us to establish a list of 38 proteins interacting with DNA adducts generated by cisplatin, oxaliplatin and satraplatin. Some of them were already known interactome members like high mobility group protein 1 (HMGB1) or the human upstream binding factor (hUBF), but we also succeeded in identifying unexpected proteins such as TOX HMG box family member 4 (TOX4), phosphatase 1 nuclear targeting subunit (PNUTS), and WD repeat-containing protein 82 (WDR82), members of a recently discovered complex. Interaction between TOX4 and platinated DNA was subsequently validated by surface plasmon resonance imaging (SPRi). These interactions highlight new cellular responses to DNA damage induced by chemotherapeutic agents.