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.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

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.     

Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis

Protein ubiquitylation is a central regulatory mechanism that controls numerous processes in plants, including hormone signaling, developmental progression, responses to biotic and abiotic challenges, protein trafficking and chromatin structure. Despite data implicating thousands of plant proteins as targets, so far only a few have been conclusively shown to be ubiquitylated in planta . Here we describe a method to isolate ubiquitin–protein conjugates from Arabidopsis that exploits a stable transgenic line expressing a synthetic poly- UBQ gene encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine. Following sequential enrichment by Ub-affinity and nickel chelate-affinity chromatography, the ubiquitylated proteins were trypsinized, separated by two-dimensional liquid chromatography, and analyzed by mass spectrometry. Our list of 54 non-redundant targets, expressed by as many as 90 possible isoforms, included those predicted by genetic studies to be ubiquitylated in plants (EIN3 and JAZ6) or shown to be ubiquitylated in other eukaryotes (ribosomal subunits, elongation factor 1α, histone H1, HSP70 and CDC48), as well as candidates whose control by the Ub/26S proteasome system is not yet appreciated. Ub attachment site(s) were resolved for a subset of these proteins, but surprisingly little sequence consensus was detected, implying that specific residues surrounding the modified lysine are not important determinants for ubiquitylation. We also identified six of the seven available lysine residues on Ub itself as Ub attachment sites, together with evidence for a branched mixed-linkage chain, suggesting that the topologies of Ub chains can be highly complex in plants. Taken together, our method provides a widely applicable strategy to define ubiquitylation in any tissue of intact plants exposed to a wide range of conditions.     

A novel mass spectrometric procedure to rapidly determine the position of O-acylated residues in the sequence of naturally occurring oligosaccharides

A novel procedure is described for the assignment of the position of O-acylated residues in the sequence of oligosaccharides. The procedure is both rapid and sensitive and complements existing carbohydrate techniques. The procedure involves the use of derivatisation, using conditions under which naturally occurring O-acyl groups are stable, and subsequent fast atom bombardment mass spectrometric analysis of the derivatised product. The derivatisation enhances and directs fragmentation providing unambiguous sequence data. The protocol described is shown to be applicable to acetyl groups and to more complex acyl groups such as long chain fatty acids.     

Challenges and opportunities in mass spectrometric analysis of proteins from dried blood spots

Dried blood spots (DBS) have been used as a clinical sample format for over 50 years, and have been analyzed for small molecules and metabolites by mass spectrometry (MS) since the early 1990s. In the meantime, MS has become the tool of choice in proteomics. Despite this obvious avenue of scientific investigation, the marriage of MS and DBS protein analysis has been comparatively recent. DBS are a potentially rich source of protein biomarkers that remain to be exploited. This article focuses on the progress made in the mass spectrometric analysis of proteins from DBS and discusses the benefits and challenges facing this emerging field.     

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.     

A comparison of the folding kinetics of a small,artificially selected DNA aptamer with those of equivalently simple naturally occurring proteins

The folding of larger proteins generally differs from the folding of similarly large nucleic acids in the number and stability of the intermediates involved. To date, however, no similar comparison has been made between the folding of smaller proteins, which typically fold without well-populated intermediates, and the folding of small, simple nucleic acids. In response, in this study, we compare the folding of a 38-base DNA aptamer with the folding of a set of equivalently simple proteins. We find that, as is true for the large majority of simple, single domain proteins, the aptamer folds through a concerted, millisecond-scale process lacking well-populated intermediates. Perhaps surprisingly, the observed folding rate falls within error of a previously described relationship between the folding kinetics of single-domain proteins and their native state topology. Likewise, similarly to single-domain proteins, the aptamer exhibits a relatively low urea-derived Tanford β, suggesting that its folding transition state is modestly ordered. In contrast to this, however, and in contrast to the behavior of proteins, ϕ-value analysis suggests that the aptamer''s folding transition state is highly ordered, a discrepancy that presumably reflects the markedly more important role that secondary structure formation plays in the folding of nucleic acids. This difference notwithstanding, the similarities that we observe between the two-state folding of single-domain proteins and the two-state folding of this similarly simple DNA presumably reflect properties that are universal in the folding of all sufficiently cooperative heteropolymers irrespective of their chemical details.     

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.