Recombinant expression of Mus musculus myoglobin

The cDNA encoding for Mus musculus myoglobin (Mb) was amplified using standard RT-PCR techniques and cloned in an appropriate bacterial expression vector. For the first time, mouse Mb was recombinantly expressed in Escherichia coli cells, BL21(DE3), and purified in sufficient amounts to carry out a preliminary characterization. As shown by mass spectrometry, the protein is found in complex with glutathione, which binds the Cys residue in the topological position E9, in the proximity of the heme group. In recombinant murine Mb, azide affinities are only slightly dependent on the Cys(E9) oxidation state. This suggests that Cys(E9) does not provide a relevant contribution for the stabilization of ligands bound to the heme iron atom. Recombinant expression of M. musculus Mb might have an important role in order to investigate the eventual involvement of Cys(E9) in the new physiological roles proposed for Mb.     

Interaction of cisplatin and analogues with a Met-rich protein site

The chaperone protein CopC from Pseudomonas syringae features high-affinity binding sites (K _D ~ 10⁻¹³ M) for both Cu^I (Met-rich) and Cu^II (His-rich). When presented with these sites in the apoprotein, electrospray ionisation mass spectrometry confirmed that cis-Pt(NH₃)₂Cl₂ (cisplatin) and the fragments [Pt^IIL]²⁺ (L is 1,2-diaminoethane, 2,2′-bipyridine) occupied the Cu^I site specifically in the 1:1 Pt–CopC adducts (purified by cation-exchange chromatography). The cis-Pt(NH₃)₂ fragment was not present in these adducts (the dominant product for cisplatin was Pt–CopC in which all original ligands were displaced), while bidentate ligands L were retained in LPt–CopC adducts. In the context of the Met-rich Cu^I pump Ctr1 as a significant entry point for cisplatin into mammalian cells, the present work confirms the ability of Met-rich sites in proteins to remove all ligands from cisplatin. It focuses attention on the potential of proteins that are part of the natural copper transport pathways to sequester the drug. These pathways are worthy of further study at the molecular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategy

The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ.     

Recent advances in the mass spectrometric profiling of bacterial lipids

Exploring the lipids of bacteria presents a predicament that may not be broadly recognized in a field dominated by the biology and biochemistry of eukaryotic — and especially, mammalian — lipids. Bacteria make multifarious metabolites that contain fatty acyl chains of unusual length and unsaturation attached to assorted headgroups, including sugars and fatty alcohols. Lipid profiling approaches developed for eukaryotic lipids often fail to detect, resolve, or identify bacterial lipids due to their wide range of polarities (including very hydrophobic species) and diverse positional and stereochemical variations. Global lipid profiling, or lipidomics, of bacteria has thus developed as a separate mission with methodological and scientific considerations tailored to the biology of these organisms. In this review, we summarize findings primarily from the last three years that exemplify recent advances and continuing challenges to learning about bacterial lipids.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Energy-dependent electrospray ionization mass spectrometric studies of mononuclear metal carbonyls

Energy-dependent electrospray ionization mass spectrometry (EDESI-MS) technique has been used for investigating the fragmentation of monomeric metal carbonyl complexes of general formula [M(CO)_x(COOMe)]⁻ (M = Cr, Fe, Mo, and W). The results show that in addition to the loss of CO, formaldehyde loss provides a pathway of fragmentation. Of all complexes investigated, the chromium complex exhibits the cleanest fragmentation. At very high voltages, the metal ions is either associated with a hydride or a methoxide ions.     

Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation

Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS³ in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.     

Human serum butyrylcholinesterase interactions with cisplatin and cyclophosphamide

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 ± 50.4 μM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 × 10⁻⁵ M⁻¹ min⁻¹, 0.063 min⁻¹, and 9.83 × 10⁻⁴ M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 ± 71.2 μM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.     

Investigation of protein-RNA interactions by mass spectrometry--Techniques and applications

Protein-RNA complexes play many important roles in diverse cellular functions. They are involved in a wide variety of different processes in growth and differentiation at the various stages of the cell cycle. As their function and catalytic activity are directly coupled to the structural arrangement of their components--proteins and ribonucleic acids--the investigation of protein-RNA interactions is of great functional and structural importance. Here we discuss the most prominent examples of protein-RNA complexes and describe some frequently used purification strategies. We present various techniques and applications of mass spectrometry to study protein-RNA complexes. We discuss the analysis of intact complexes as well as proteomics-based and crosslinking-based approaches in which proteins are cleaved into smaller peptides. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Protein identification by in-gel digestion and mass spectrometric analysis

This protocol details a method for the identification of proteins that have been separated by gel electrophoresis. In-gel digestion of the protein bands with trypsin followed by quadrupole ion-trap or other triple quadrupole mass spectrometry techniques is described. The proteins can be identified by database searching of the mass fingerprint of the intact peptides and of the characteristic fragment masses produced by tandem mass spectrometry.     

DNA and glutathione interactions in cell-free media of asymmetric platinum(II) complexes cis- and trans-[PtCl2(isopropylamine)(1-methylimidazole)]: relations to their different antitumor effects

The global modification of mammalian and plasmid DNAs by the novel platinum compounds cis-[PtCl₂(isopropylamine)(1-methylimidazole)] and trans-[PtCl₂(isopropylamine)(1-methylimidazole)] and the reactivity of these compounds with reduced glutathione (GSH) were investigated in cell-free media using various biochemical and biophysical methods. Earlier cytotoxicity studies had revealed that the replacement of the NH₃ groups in cisplatin by the azole and isopropylamine ligands lowers the activity of cisplatin in both sensitive and resistant cell lines. The results of the present work show that this replacement does not considerably affect the DNA modifications by this drug, recognition of these modifications by HMGB1 protein, their repair, and reactivity of the platinum complex with GSH. These results were interpreted to mean that the reduced activity of this analog of cisplatin in tumor cell lines is due to factors that do not operate at the level of the target DNA. In contrast, earlier studies had shown that the replacement of the NH₃ groups in the clinically ineffective trans isomer (transplatin) by the azole and isopropylamine ligands results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin, which is distinctly different from that of cisplatin, but does not affect reactivity with GSH. Hence, the results of the present work are consistent with the view and support the hypothesis systematically tested by us and others that platinum drugs that bind to DNA in a fundamentally different manner from that of conventional cisplatin may have altered pharmacological properties.     

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.     

Comprehensive two-dimensional liquid chromatography mass spectrometric profiling of the rat hippocampal proteome

In this study, we performed the first high‐throughput and comprehensive proteomic profiling of the rat hippocampal proteome. Using a combination of 2‐D LC‐MS and data analysis with the Rosetta Elucidator^® system, we identified 1340 unique proteins. Functional classification showed that most of these were associated with synaptic function and comprised a high proportion of phosphorylated proteins and analytically challenging classes of membrane proteins such as ion channel receptor subunits.     

Direct mass spectrometric measurement of gases in peat cores

Abstract Dissolved gas concentrations (O₂, CH₄, CO₂) in peat cores were monitored simultaneously using a fine (1.56 mm diameter) membrane inlet probe connected to a quadrupole mass spectrometer. This technique allows direct measurements at specific locations within the sample with minimal disturbance. Detailed gas profiles in completely waterlogged peat samples (hollows) and samples in which the water table was several cm below the vegetation surface (hummocks) were compared. The depth of the water table played a central role in the distribution of gases. In a hollow, oxygen was present (90 μM) at the surface but was not detectable (<0.5 μM) at depths greater than 2 cm. Concentrations of CH₄ and CO₂ increased from 6 and 300 μM respectively at the surface to maxima of 450 and 3900 μM at 13 cm depth. At a hummock, O₂ and CO₂ were present above the water table but CH₄ was not detectable. CH₄ was measurable 2 cm below the water table. Both CH₄ and CO₂ concentrations increased with depth but maxima were not attained in the sampled cores.     

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.     

A mass spectrometric analysis of the water-splitting reaction

Earlier mass spectrometric measurements, in which oxygen evolution was measured following short saturating light flashes, indicated that with a time resolution of about 30 s no form of bound water and/or an oxidation product exists up to the redox state S₃ of the oxygen evolving center (R. Radmer and O. Ollinger, 1986, FEBS Lett 195: 285–289; K.P. Bader, P. Thibault and G.H. Schmid, 1987, Biochim Biophys Acta 893: 564–571). In the present study, isotope exchange experiments with H₂ ¹⁸O were performed under different experimental conditions. We found: a) the isotope exchange pattern is virtually the same at both pH 6.0 and 7.8, although marked structural changes of the PS II donor side are inferred to take place within this pH-range (Renger G., Messinger J. and Wacker U., 1992, Research in Photosynthesis, II: 329–332); b) injection of H₂ ¹⁸O at about 0°C gives rise to mass ratios of the evolved oxygen which markedly deviate from the theoretically expected values of complete isotope scrambling; and c) rapid injection of H₂ ¹⁸O into samples with high population of S₁ and S₂ and subsequent illumination with three and two flashes, respectively, spaced by a dark time of only 1.5 ms lead to similar ¹⁸O-labeling of the evolved oxygen. Based on the published data on the interaction with redox active amines, possible pathways of substrate exchange in the water oxidase are discussed.Abbreviations atom fraction of ¹⁸O - PS II Photosystem II - S_i redox states of the water oxidase - Y_z redox active tyrosine of polypeptide D1     

How mass spectrometric approaches applied to bacterial identification have revolutionized the study of human gut microbiota

Introduction: Describing the human hut gut microbiota is one the most exciting challenges of the 21^st century. Currently, high-throughput sequencing methods are considered as the gold standard for this purpose, however, they suffer from several drawbacks, including their inability to detect minority populations. The advent of mass-spectrometric (MS) approaches to identify cultured bacteria in clinical microbiology enabled the creation of the culturomics approach, which aims to establish a comprehensive repertoire of cultured prokaryotes from human specimens using extensive culture conditions.

Areas covered: This review first underlines how mass spectrometric approaches have revolutionized clinical microbiology. It then highlights the contribution of MS-based methods to culturomics studies, paying particular attention to the extension of the human gut microbiota repertoire through the discovery of new bacterial species.

Expert commentary: MS-based approaches have enabled cultivation methods to be resuscitated to study the human gut microbiota and thus to fill in the blanks left by high-throughput sequencing methods in terms of culturing minority populations. Continued efforts to recover new taxa using culture methods, combined with their rapid implementation in genomic databases, would allow for an exhaustive analysis of the gut microbiota through the use of a comprehensive approach.     

Electrospray mass spectrometric decomposition of some glucuronic acid-containing flavonoid diglycosides

Mass spectrometric fragmentation patterns of herbacetin 3-O-glucopyranoside-8-O-glucuronopyranoside (1), gossypetin 3-O-glucopyranoside-8-O-glucuronopyranoside (2) and takakin 7-O-glucopyranoside-8-O-glucuronopyranoside (3) were elucidated from mass spectra obtained with electrospray ionisation. The usefulness of the fragmentation patterns observed in the positive and negative mode for structural elucidation of the studied compounds is discussed. The fragmentation of 3 was substantially different from 1 and 2, especially in the negative mode. In order to explain these differences, theoretical calculations were performed.     

In vivo detection of secreted proteins from wounded skin using capillary ultrafiltration probes and mass spectrometric proteomics

The identification of in vivo secreted proteins is a major challenge in systems biology. Here we report a novel technique using capillary ultrafiltration (CUF) probes to identify the secreted proteins involved in wound healing. CUF probes, which use semipermeable membrane hollow fibers to continuously capture secreted proteins, were used to sample skin wound fluids. To identify low-abundance proteins, we digested the CUF probe-collected wound fluid with trypsin and then directly subjected it to MS without using 2-DE separation. Two protein fragments with masses of 1565.7 and 1694.8 Da were identified by MS as peptides of thymosin beta10 and beta4, respectively. This is the first identification of thymosin beta10 as an in vivo constituent of the skin wound fluid. The LKKTETQ peptide, a common actin-binding domain of thymosin beta4 and beta10, significantly enhanced skin wound healing in vitro and in vivo. Our data suggest that the enhancement of wound healing by LKKTETQ may be mediated by purinergic receptors. The technique of using CUF probes linked to mass spectrometric proteomics represents a powerful method to identify in vivo secreted proteins, and may be applicable for identification of proteins relevant in various human diseases.