Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.     

Effect of chronic morphine exposure on the synaptic plasma-membrane subproteome of rats: a quantitative protein profiling study based on isotope-coded affinity tags and liquid chromatography/mass spectrometry

The effect of chronic morphine exposure on the synaptic plasma-membrane subproteome in rats was studied by the isotope-coded affinity tag (ICAT) method coupled with capillary reversed-phase liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. ICAT-labeled tryptic peptides of synaptic membrane proteins were successfully identified using tandem mass spectrometry in conjunction with protein database searching. Several important synaptic plasma-membrane proteins displayed significant regulation changes as a result of chronic morphine exposure in vivo. In particular, an integral membrane protein Na(+)/K+ ATPase (alpha-subunit) involved in regulation of the cell membrane potential by controlling sodium and potassium ion permeability was downregulated by 39 +/- 2%. This result was in excellent agreement with the reduction in electrogenic Na+, K+ pumping due to about 40% downregulation of Na(+)/K+ ATPase alpha3-isoform in myenteric S-neurons of morphine-exposed guinea-pigs measured by others via immunohistochemistry. The decrease in the abundance of non-erythroid alpha II-spectrin in the synaptic plasma-membrane fraction was also observed, which was hypothetically associated with the breakdown of the protein due to the upregulation of the proteolytic enzyme caspase-3 upon chronic morphine exposure.     

Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral "fingerprint" generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

Microfluidic electrocapture interfaced with electrospray mass spectrometry

Microfluidic electrocapture of peptides and proteins in an inert capillary with electric contacts via conductive membranes is useful for sample handling before mass spectrometry. The use of electrocapture has already been demonstrated for sample clean-up, pre-concentration, chemical modification and peptide separation, all without the need for supporting gels or chemical binding. This method allows multiple micro-reactions, extensive peptide separations and work with membrane proteins from detergent-solubilized samples. Until now, electrocapture has utilized MALDI mass spectrometry, but here we demonstrate that it can be interfaced with electrospray ionization and hence with on-line mass spectrometric analysis of peptides separated from protein digests. These applications combined with the present on-line approach show electrocapture to be a versatile technology with great potential.     

Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been used in conjunction with time-of-flight (TOF) and quadrupole ion trap (IT) mass spectrometry, respectively, to analyze various cytolysin proteins isolated from the sea anemone Stichodactyla helianthus and digested by the protease trypsin. By employing different ionization methods, the subsequent changes in ionization selectivity for the peptides in the digested protein samples resulted in ion abundance variation reflected in the mass spectra. Upon investigation of this variation generated by the two ionization processes, it has been shown in this study that enhanced protein coverage (e.g., >95% for cytolysin III) can be achieved. Additionally, capillary and microbore reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ESI mass spectrometry (MS) as well as flow injection analysis by nanoflow ESI-MS afforded the necessary limit of detection (LOD) for detailed structural information of the cytolysin proteins by tandem mass spectrometry (MS/MS) methods. It can be concluded that cytolysins II and III correspond to sticholysins I and II, that "cytolysin I" is a mixture of modified forms of cytolysins II and III, and that "cytolysin IV" is an incompletely processed precursor of cytolysin III.     

Characterization of multi-phosphopeptides by muHPLC-ESI-MS/MS with alkaline phosphatase treatment

The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by muHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of beta-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by muHPLC-ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.     

Targeted comparative proteomics by liquid chromatography/matrix-assisted laser desorption/ionization triple-quadrupole mass spectrometry

Here we report the first application of a matrix-assisted laser desorption/ionization (MALDI) triple-quadrupole mass spectrometer for targeted proteomics. Employing an amine-specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n = 5) and a detection limit of 14 amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two-dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D-LC/ESI)-MS/MS. Using LC/MALDI-MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7 +/- 0.8.     

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

纳升电喷雾萃取电离质谱快速测定人参皂苷

以自行研制的纳升电喷雾萃取电离源（NanoEESI）为基础,建立了在无需色谱分离条件下快速测定人参皂苷的质谱分析方法,用NanoEESI串联质谱分析鉴定了人参中的多种人参皂苷,并将该方法用于人参、桔梗和商陆的快速鉴定。实验表明,人参中的皂苷易于在电离过程中结合钠离子,并形成[M+Na]+正离子从而被检测分析,而桔梗和商陆等样品中不含人参皂苷成份,在串联质谱中也没有相应的特征碎片离子,从而达到鉴别中药材真伪的目的。本方法简便、快速、灵敏、特征性强、重现性好,对道地中药材快速鉴定提供了思路,具有很好的实用价值。     

Identification of phosphorylation sites in phosphopeptides by positive and negative mode electrospray ionization-tandem mass spectrometry

A series of synthetic mono- and diphosphorylated peptides has been analyzed by positive and negative mode electrospray ionization-tandem mass spectrometry. The synthetic peptides are serine- and threonine-phosphorylated analogs of proteolytic fragments from the C-terminal region of rhodopsin. Use of positive and negative modes of electrospray ionization to produce ions for tandem mass spectrometry via low energy collision-induced dissociation was explored. For some of the peptides, the complementary use of experimental results allowed determination of the phosphorylation sites when either mode alone gave incomplete information. Other peptides, however, gave negative ion spectra not interpretable in terms of backbone cleavages. However, use of positive ion tandem mass spectrometry of different charge state precursor ions gave sufficient information in most cases to assign sites of phosphorylation. These results illustrate the utility of obtaining complementary information by tandem mass spectrometry by using precursor ions of different charge polarity or number.     

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.     

Biomedical and biological mass spectrometry.

This review focuses on biological and biomedical mass spectrometry, and covers a selection of publications in this area included in the MEDLINE database for the period 1987-2001. Over the last 15 years, biological and biomedical mass spectrometry has progressed out of all recognition. The development of soft ionization methods, such as electrospray ionization and matrix-assisted laser desorption ionization, has mainly contributed to the remarkable progress, because they can easily produce gas-phase ions of large, polar, and thermally labile biomolecules, such as proteins, peptides, nucleic acids and others. The innovations of ionization methods have led to remarkable progress in mass spectrometric technology and in biochemistry, biotechnology and molecular biology research. In addition, mass spectrometry is one of the powerful and effective technologies for drug discovery and development. It is applicable to studies on structural determination, drug metabolism, including pharmacokinetics and toxicokinetics, and de novo drug discovery by applying post-genomic approarches. In the present review, the innovative soft ionization methods are first discussed along with their features. Also, the characteristics of the mass spectrometers which are active in the biological and biomedical research fields are also described. In addition, examples of the applications of biological and biomedical mass spectrometry are provided.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

Tandem mass spectrometry of model peptides modified with trans-2-hexenal, a product of lipid peroxidation

Small molecules formed during lipid peroxidation can react with the basic groups in proteins through different mechanisms. Recently, substituted pyridinium moieties were observed during in vitro incubations of lysine-containing peptides with 2-alkenals. To explore the dissociation behavior of peptides with pyridinium-derivatized lysine residues, the peptide ions created through either matrix-assisted laser desorption/ionization or electrospray ionization were studied with tandem mass spectrometry. The permanently charged pyridinium ions fragment primarily through the charge-remote processes. Under high energy collision-induced dissociation, a number of diagnostic ions were observed that could potentially be used to identify modified residues in proteins. The origins of these ions were studied using deuterium exchange and higher-order mass spectrometry experiments using an ion trap instrument. Rational structures for these ions are proposed.     

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein–protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide “molecular pictures”, which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.     

Deep‐ultraviolet laser ablation electrospray ionization mass spectrometry

A 193‐nm wavelength deep ultraviolet laser was used for ambient laser ablation electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer laser was used to ablate solid samples, and the resulting plume of the desorbed material merged with charged electrospray droplets to form ions that were detected with a quadrupole time‐of‐flight mass spectrometer. Solutions containing peptide and protein standards up to 66‐kDa molecular weight were deposited on a metal target, dried, and analyzed. No fragmentation was observed from peptides and proteins as well as from the more easily fragmented vitamin B₁₂ molecule. The mass spectra contained peaks from multiply charged ions that were identical to conventional electrospray. Deep UV laser ablation of tissue allowed detection of lipids from untreated tissue. The mechanism of ionization is postulated to involve absorption of laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or by residual water in the sample.     

Structural analysis of naphthoquinone protein adducts with liquid chromatography/tandem mass spectrometry and the scoring algorithm for spectral analysis (SALSA)

The relative reactivities of various naphthoquinone isomers (1,4-, 1,2- and 2-methyl-1,4-naphthoquinone) to two test proteins, apomyoglobin and human hemoglobin, were evaluated via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The structural characterization of the resulting adducts was also obtained by LC/ESI-MS analysis of the intact proteins. The reactive sites of apomyoglobin and human hemoglobin with 1,4-naphthoquinone and 1,2-naphthoquinone were also identified through characterization of adducted tryptic peptides by use of high-pressure liquid chromatography/electrospray ionization with tandem mass spectrometry (HPLC/ESI-MS/MS), TurboSEQUEST, and the scoring algorithm for spectral analysis (SALSA). Four adducted peptides, which were formed by nucleophilic addition of a lysine amino acid residue to 1,4-naphthoquinone, were also identified, as was an adducted peptide from incubation of 1,2-naphthoquinone with apomyoglobin. In the case of incubation of human hemoglobin with the two naphthoquinones, two adducted peptides were identified from the N-terminal valine modification of the alpha and beta chains of human hemoglobin. The adducted protein formation may imply that naphthalene produces its in vivo toxicity through 1,2- and 1,4-naphthoquinone metabolites reacting with biomolecular proteins.     

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states. The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.