Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.     

Mass spectrometric analysis of cross-linking sites for the structure of proteins and protein complexes

Mass spectrometry has become the method of choice to detect and quantify the minute amounts of proteins at the genomic scale. It has recently been adopted for three dimensional structure analyses of proteins or protein complexes by chemically cross-linking their intact forms and analyzing the cross-linked pieces after digestion. This highlight provides an overview of the technology with a focus on advances in the last two years. This cross-linking mass spectrometry has a great potential to become a powerful tool to supplement current X-ray and NMR method of protein structure analysis.     

MALDI-TOF mass spectrometry of naturally occurring mixtures of monorhamnolipids and dirhamnolipids

MALDI-TOFMS approaches have been developed for high-throughput screening of naturally occurring mixtures of rhamnolipids from Pseudomonas spp. Monorhamnolipids and dirhamnolipids are readily distinguished by characteristic molecular adduct ions, [M+Na]⁺ and [M−H+Na₂]⁺, with variously acylated rhamnolipids differing by 28 mu. Following proton-deuterium exchange, deuterated [M+Na−4¹H+4²H]⁺ and [M+Na−6¹H+6²H]⁺ ions are observed for the monorhamnolipids and dirhamnolipids, respectively, which allows rapid identification of these molecules. The described approach has been validated by compositional analysis using GC/MS, fractionation by RPHPLC, and analysis by 1D and 2D NMR spectroscopy. MALDI-TOFMS analysis allows the rapid screening of variously acylated rhamnolipids, and has potential for selective identification of new surfactants from microbial strains.     

Wavelike distributions of infections by an introduced and naturally occurring root pathogen along wheat roots

Previously, we showed that copiotrophic and oligotrophic bacteria fluctuate as moving waves along roots. These waves probably originate as a result of growth and death cycles at any location where a moving nutrient source passed. In this study, we placed sclerotia of Rhizoctonia solani AG8 along growing roots of wheat and showed that the proportions of root sections from which R. solani was isolated fluctuated with distance from the root tip. Similarly, proportions of root sections from which naturally occurring Pythium spp. were isolated fluctuated with distance from the root tip. Fourier analysis showed that these fluctuations constituted significant waves. Cross-correlation analyses demonstrated that there were negative correlations between R. solani infections and colony forming units of copiotrophic bacteria at the time of inoculation at the same locations on the root (lag = 0 cm), indicating that infection by R. solani could have been inhibited by these bacteria. There was a positive correlation between Pythium infections and copiotrophic bacteria at a lag of 6 cm along the roots. It therefore appears that Pythium infection took place shortly after the initial peak in copiotrophic bacteria following the passage of the root tip.     

Computer-assisted, high-performance liquid chromatography with mass spectrometric detection for the analysis of coumarins in Peucedanum palustre and Angelica archangelica

A reversed-phase HPLC method with atmospheric pressure chemical ionisation MS detection has been developed for the separation and identification of coumarins in plants of Peucedanum palustre L. (Moench) and Angelica archangelica (L.) var. archangelica. The Turbo Method Development program was utilised to optimise the mobile phase with two organic solvents (acetonitrile and methanol) and two aqueous solutions (1.0% formic acid and 10 mM ammonium acetate). Optimisation of the solvent gradients for the method was performed with the aid of the DryLab program. Analyses were carried out using a Phenomenex Prodigy RP C18 column. Fifty-two peaks (14 of which were associated with coumarins) were separated in 30 min from extracts of P. palustre, and 48 peaks (15 associated with coumarins) from extracts of A. archangelica. A total of 21 different coumarin-type compounds were identified in the aerial and the underground parts of the title plants. Isopimpinellin and pimpinellin were found for the first time in P. palustre and were identified by comparison of retention times and MS data obtained following the analysis of pure standards. This is the first report of the coumarin composition of the umbels of P. palustre.     

Probing cysteine reactivity in proteins by mass spectrometric EC-tagging

The on-line electrochemical tagging (EC-tagging) of cysteine residues in proteins during mass spectrometry is studied to probe the cysteine environment. Benzoquinone probes electrogenerated at a microspray electrode react with the thiol functions of the proteins within a microchannel and the products are analyzed by mass spectrometry. The fundamentals of the technique are discussed, with a focus on the kinetic aspects. The EC-tagging efficiency of the cysteine residues in proteins is used to probe their environment. Experiments with unmodified proteins and their chemically reduced forms highlight the strong effect of the cysteine site reactivity on the tagging efficiencies. This study highlights relevant parameters for such on-line electrochemical derivatization/MS detection strategies.     

Differential proteome analysis and mass spectrometric characterization of germ line development-related proteins of Caenorhabditis elegans

Proteome maps and differences of protein patterns of the synchronized larval stage L4 of the temperature-sensitive Caenorhabditis elegans (C. elegans) glp-1 mutant (e2144ts) were investigated after cultivation at 15 degrees C (developing a normal phenotype) or 25 degrees C (developing a mutated phenotype) by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. From the 183 identified protein spots six proteins were found differently expressed. The Vit-6 vitellogenin (CE28594), the hypothetical 17.2 protein (CE25224), the hypothetical 17.4 protein (CE16999), and the heat shock protein 16 kDa (CE14249) were more abundant when growing worm cultures at 25 degrees C. By contrast, the nucleoside diphosphate kinase (CE09650) was found increased at 15 degrees C. Most notably, the eukariotic initiation factor 5A-1 (CE00503), highly abundant at 15 degrees C, was not present in cultures grown at 25 degrees C. Its absence at 25 degrees C can not be attributed to lack of the enzymatic machinery that is necessary for hypusinylation. Instead, a direct downstream effect of the lack of functionality of GLP-1 may cause the expression of this protein. The yolk proteins 115 kDa and 88 kDa were attributed by mass spectrometric protein structure analysis as C-terminal and N-terminal fragments of the Vit-6 vitellogin protein (CE28594), respectively. The cleavage site between both derivatives was located between R764 and A768. A conflict in the database sequences at amino acid positions 1622 and 1623 of vitellogenin-6 was solved by mass spectrometric sequence analysis. The combination of 2-DE with mass spectrometry enabled the identification of mutation-associated differences on somatic gonadal cell and germ line cell development-associated proteins.     

Direct analysis of protein complexes using mass spectrometry.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.     

Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds

The disulfide bond bridge is an important post-translational modification for proteins. This study presents a structural analysis of biologically active peptides and proteins containing disulfide bonds using electrochemistry (EC) online combined with desorption electrospray ionization mass spectrometry (DESI-MS), in which the sample undergoes electrolytic disulfide cleavage in an electrochemical flow cell followed by MS detection. Using this EC/DESI-MS method, the disulfide-containing peptides can be quickly identified from enzymatic digestion mixtures, simply based on the abrupt decrease in their relative ion abundances after electrolysis. Peptide mass mapping and tandem MS analysis of the ions of the resulting free peptide chains can possibly establish the disulfide linkage pattern and sequence the precursor peptides. In this regard, the method provides much more chemical information than previous analogous electrochemical analyses. In addition, derivatization of thiols by selective selenamide reagents is useful for easy recognition of reduced peptide ions and the number of their free thiols. Furthermore, electrolytic reduction of proteins (e.g., α-lactalbumin) leads to increased charges on the detected protein ions, revealing the role of disulfide bonds on maintaining protein conformation. This electrochemical mass spectrometric method is fast (completed in few minutes) and does not need chemical reductants, potentially having valuable applications in proteomics research.     

Interactions among phytophagous mites, and introduced and naturally occurring predatory mites, on strawberry in the UK

In choice test experiments on strawberry leaf disc arenas the phytoseiid mites Neoseiulus californicus and N. cucumeris were more effective than Typhlodromus pyri as predators of the phytophagous mites Tetranychus urticae and Phytonemus pallidus. There were no preferences shown for either prey by any of these predators. In multiple predator leaf disc experiments both Phytoseiulus persimilis and N. cucumeris significantly reduced numbers of T. urticae eggs and active stages; this effect was seen when the two species were present alone or in combination with other predator species. Neoseiulus californicus was less effective at reducing T. urticae numbers, and T. pyri was not effective; no interaction between predator species was detected in these experiments. When T. urticae alone was present as prey on potted plants, P. persimilis and N. californicus were the only phytoseiids to significantly reduce T. urticae numbers. These two predator species provided effective control of T. urticae when P. pallidus was also present; however, none of the predators reduced numbers of P. pallidus. There were no significant negative interactions when different species of predators were present together on these potted plants. In field experiments, releases of both P. persimilis and N. cucumeris significantly reduced T. urticae numbers. However, there was a significant interaction between these predator species, leading to poorer control of T. urticae when both species were released together. These results show the importance of conducting predator/prey feeding tests at different spatial scales.     

Activation of protein kinase C by naturally occurring ether-linked diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.     

Hidden localization motifs: naturally occurring peroxisomal targeting signals in non-peroxisomal proteins

Background

Can sequence segments coding for subcellular targeting or for posttranslational modifications occur in proteins that are not substrates in either of these processes? Although considerable effort has been invested in achieving low false-positive prediction rates, even accurate sequence-analysis tools for the recognition of these motifs generate a small but noticeable number of protein hits that lack the appropriate biological context but cannot be rationalized as false positives.

Results

We show that the carboxyl termini of a set of definitely non-peroxisomal proteins with predicted peroxisomal targeting signals interact with the peroxisomal matrix protein receptor peroxin 5 (PEX5) in a yeast two-hybrid test. Moreover, we show that examples of these proteins - chicken lysozyme, human tyrosinase and the yeast mitochondrial ribosomal protein L2 (encoded by MRP7) - are imported into peroxisomes in vivo if their original sorting signals are disguised. We also show that even prokaryotic proteins can contain peroxisomal targeting sequences.

Conclusions

Thus, functional localization signals can evolve in unrelated protein sequences as a result of neutral mutations, and subcellular targeting is hierarchically organized, with signal accessibility playing a decisive role. The occurrence of silent functional motifs in unrelated proteins is important for the development of sequence-based function prediction tools and the interpretation of their results. Silent functional signals have the potential to acquire importance in future evolutionary scenarios and in pathological conditions.     

Increased thermal stability of proteins in the presence of naturally occurring osmolytes.

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)