Mass Spectrometry Strategy Research Articles

Using Proteomic Strategies for Sequencing and Post-Translational Modifications Assignment of Antigen-5, a Major Allergen from the Venom of the Social Wasp Polybia paulista

Antigen-5 is one of the major allergens identified in wasp venoms, and despite the fact that its biological function is still unknown, many studies have demonstrated its allergenicity. In this study, the biochemical and structural characterization of antigen-5 from the venom of the social wasp Polybia paulista are reported. A gel-based mass spectrometry strategy with CID fragmentation methods and classical protocols of protein chemistry, which included N- and C-terminal sequencing, were used to assign the complete sequence and determine the presence/location of the post-translational modifications (PTMs) of this protein. Six different isoforms of antigen-5 were identified in the crude venom of P. paulista ; the most abundant, which corresponds to the intact form of this protein, was recognized by the pool of human specific-IgE. This protein was extensively sequenced through CID mass spectrometry, and a series of PTMs were observed such as hydroxylation, phosphorylation, and glycosylation. Sequence data revealed that this protein has 59.3-93.7% identity with antigen-5 proteins from other known vespid venoms. The molecular model of P. paulista antigen-5 shows that this protein has three α-helices, one 310 helix, and four β-sheets covering 28 and 17.9% of the sequence, respectively. The identification and characterization of allergenic compounds is essential for the development of advanced component-resolved allergy diagnostics and treatment.

Evaluating the Conformation and Binding Interface of Cap-Binding Proteins and Complexes via Ultraviolet Photodissociation Mass Spectrometry

We report the structural analysis of cap-binding proteins using a chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating solvent accessibility of proteins. Our methodology utilized a chromogenic probe (NN) to probe the exposed amine residues of wheat eukaryotic translation initiation factor 4E (eIF4E), eIF4E in complex with a fragment of eIF4G ("mini-eIF4F"), eIF4E in complex with full length eIF4G, and the plant specific cap-binding protein, eIFiso4E. Structural changes of eIF4E in the absence and presence of excess dithiothreitol and in complex with a fragment of eIF4G or full-length eIF4G are mapped. The results indicate that there are particular lysine residues whose environment changes in the presence of dithiothreitol or eIF4G, suggesting that changes in the structure of eIF4E are occurring. On the basis of the crystal structure of wheat eIF4E and a constructed homology model of the structure for eIFiso4E, the reactivities of lysines in each protein are rationalized. Our results suggest that chemical probe/UVPD mass spectrometry can successfully predict dynamic structural changes in solution that are consistent with known crystal structures. Our findings reveal that the binding of m(7)GTP to eIF4E and eIFiso4E appears to be dependent on the redox state of a pair of cysteines near the m(7)GTP binding site. In addition, tertiary structural changes of eIF4E initiated by the formation of a complex containing a fragment of eIF4G and eIF4E were observed.

A new ultrahigh performance liquid chromatography with diode array detection coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry analytical strategy for fast analysis and improved characterization of phenolic compounds in apple products

A new, rapid, selective and sensitive ultrahigh performance liquid chromatography with diode array detection coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (UHPLC-DAD–ESI-Q-ToF-MS) strategy using automatic and simultaneous acquisition of exact mass at high and low collision energy, MSE, has been developed to obtain polyphenolic profile of apples, apple pomace and apple juice from Asturian cider apples in a single run injection of 22min. MSE spectral data acquisition overcomes chromatographic co-elution problems, performing simultaneous collection of precursor ions as well as other ions produced as a result of their fragmentation, which allows resolving complex spectra from mixtures of precursor ions in an unsupervised way and eases their interpretation. Using this technique, 52 phenolic compounds of five different classes were readily characterized in these apple extracts in both positive and negative ionization modes. The spectral data for phenolic compounds obtained using this acquisition mode are comparable to those obtained by conventional LC–MS/MS as exemplified in this work. Among the 52 phenolic compounds identified in this work, 2 dihydrochalcones and 3 flavonols have been tentatively identified for the first time in apple products. Moreover, 2 flavanols, 4 dihydrochalcones, 9 hydroxycinnamic acids and 4 flavonols had not been previously reported in apple by ToF analysis to our knowledge.

Affinity Purification Strategies for Proteomic Analysis of Transcription Factor Complexes

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein–protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

Chromogenic Chemical Probe for Protein Structural Characterization via Ultraviolet Photodissociation Mass Spectrometry

A chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating structures of proteins and protein complexes is reported, as demonstrated for lysozyme and beta-lactoglobulin with and without bound ligands. The chemical probe, NN, incorporates a UV chromophore that endows peptides with high cross sections at 351 nm, a wavelength not absorbed by unmodified peptides. Thus, NN-modified peptides can readily be differentiated from nonmodified peptides in complex tryptic digests created upon proteolysis of proteins after their exposure to the NN chemical probe. The NN chemical probe also affords two diagnostic reporter ions detected upon UVPD of the NN-modified peptide that provides a facile method for the identification of NN peptides within complex mixtures. Quantitation of the modified and unmodified peptides allows estimation of the surface accessibilities of lysine residues based on their relative reactivities with the NN chemical probe.

Mass spectrometry strategies for clinical metabolomics and lipidomics in psychiatry, neurology, and neuro-oncology.

Metabolomics research has the potential to provide biomarkers for the detection of disease, for subtyping complex disease populations, for monitoring disease progression and therapy, and for defining new molecular targets for therapeutic intervention. These potentials are far from being realized because of a number of technical, conceptual, financial, and bioinformatics issues. Mass spectrometry provides analytical platforms that address the technical barriers to success in metabolomics research; however, the limited commercial availability of analytical and stable isotope standards has created a bottleneck for the absolute quantitation of a number of metabolites. Conceptual and financial factors contribute to the generation of statistically under-powered clinical studies, whereas bioinformatics issues result in the publication of a large number of unidentified metabolites. The path forward in this field involves targeted metabolomics analyses of large control and patient populations to define both the normal range of a defined metabolite and the potential heterogeneity (eg, bimodal) in complex patient populations. This approach requires that metabolomics research groups, in addition to developing a number of analytical platforms, build sufficient chemistry resources to supply the analytical standards required for absolute metabolite quantitation. Examples of metabolomics evaluations of sulfur amino-acid metabolism in psychiatry, neurology, and neuro-oncology and of lipidomics in neurology will be reviewed.

Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography–Tandem Mass Spectrometry Workflows

Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated.

Drafting the CLN3 Protein Interactome in SH-SY5Y Human Neuroblastoma Cells: A Label-free Quantitative Proteomics Approach

Neuronal ceroid lipofuscinoses (NCL) are the most common inherited progressive encephalopathies of childhood. One of the most prevalent forms of NCL, Juvenile neuronal ceroid lipofuscinosis (JNCL) or CLN3 disease (OMIM: 204200), is caused by mutations in the CLN3 gene on chromosome 16p12.1. Despite progress in the NCL field, the primary function of ceroid-lipofuscinosis neuronal protein 3 (CLN3) remains elusive. In this study, we aimed to clarify the role of human CLN3 in the brain by identifying CLN3-associated proteins using a Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS) strategy combined with Significance Analysis of Interactome (SAINT). Human SH-SY5Y-NTAP-CLN3 stable cells were used to isolate native protein complexes for subsequent TAP-MS. Bioinformatic analyses of isolated complexes yielded 58 CLN3 interacting partners (IP) including 42 novel CLN3 IP, as well as 16 CLN3 high confidence interacting partners (HCIP) previously identified in another high-throughput study by Behrends et al., 2010. Moreover, 31 IP of ceroid-lipofuscinosis neuronal protein 5 (CLN5) were identified (18 of which were in common with the CLN3 bait). Our findings support previously suggested involvement of CLN3 in transmembrane transport, lipid homeostasis and neuronal excitability, as well as link it to G-protein signaling and protein folding/sorting in the ER.

Combined application of targeted and untargeted proteomics identifies distinct metabolic alterations in the tetraacetylphytosphingosine (TAPS) producing yeast Wickerhamomyces ciferrii

The Wickerhamomyces ciferrii strain NRRL Y-1031 F-60-10A is a well-known producer of tetraacetylphytosphingosine (TAPS) and used for the biotechnological production of sphingolipids and ceramides. It was our aim to gain new biological insights into the sphingolipid metabolism by employing a dual platform mass spectrometry strategy. The first step comprised metabolic (15)N-labeling in combination with label-free proteomics using high resolution LTQ Orbitrap mass spectrometry. Then selected reaction monitoring tandem mass spectrometry served for the targeted quantification of sphingoid base biosynthesis enzymes. The non-producer strain NRRL Y-1031-27 served as a reference strain. In total, 1697 proteins were identified, and 123 enzymes of main metabolic pathways were observed as differentially expressed. Major findings were: 1) the likely presence of higher carbon flux in NRRL Y-1031 F-60-10A, resulting in higher availability of pyruvate and acetyl-CoA; 2) indications of oleaginous yeast-like behavior in NRRL Y-1031 F-60-10A; and 3) approx. 2-fold higher abundance of eight sphingolipid biosynthesis enzymes in NRRL Y-1031 F-60-10A. Taken together, in NRRL Y-1031 F-60-10A, the levels of glycolytic enzymes apparently gear carbon flux towards higher acetyl-CoA synthesis rates, while simultaneously reducing protein biosynthesis in the process. Thereby, C2 building blocks for acyl-moieties and downstream sphingoid base acetylation are provided to maintain TAPS production. First quantitative proteome study for a phytosphingosine-producing yeast.

Analysis of Proteome Profile in Germinating Soybean Seed, and Its Comparison with Rice Showing the Styles of Reserves Mobilization in Different Crops

BackgroundSeed germination is a complex physiological process during which mobilization of nutrient reserves happens. In different crops, this event might be mediated by different regulatory and metabolic pathways. Proteome profiling has been proved to be an efficient way that can help us to construct these pathways. However, no such studies have been performed in soybean germinating seeds up to date.ResultsProteome profiling was conducted through one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy in the germinating seeds of soybean (glycine max). Comprehensive comparisons were also carried out between rice and soybean germinating seeds. 764 proteins belonging to 14 functional groups were identified and metabolism related proteins were the largest group. Deep analyses of the proteins and pathways showed that lipids were degraded through lipoxygenase dependent pathway and proteins were degraded through both protease and 26S proteosome system, and the lipoxygenase could also help to remove the reactive oxygen species during the rapid mobilization of reserves of soybean germinating seeds. The differences between rice and soybean germinating seeds proteome profiles indicate that each crop species has distinct mechanism for reserves mobilization during germination. Different reserves could be converted into starches before they are totally utilized during the germination in different crops seeds.ConclusionsThis study is the first comprehensive analysis of proteome profile in germinating soybean seeds to date. The data presented in this paper will improve our understanding of the physiological and biochemical status in the imbibed soybean seeds just prior to germination. Comparison of the protein profile with that of germinating rice seeds gives us new insights on mobilization of nutrient reserves during the germination of crops seeds.

Profiling of diferulates (plant cell wall cross-linkers) using ultrahigh-performance liquid chromatography-tandem mass spectrometry

Recalcitrance of grasses to enzymatic digestion arises to a significant degree from a complex array of phenolic crosslinks between cell wall polysaccharide chains that inhibit their conversion to biofuels and lower their nutritive value for animal feed applications. Polysaccharide esters of ferulic acid are abundant in plant cell walls. Crosslinks between polysaccharides are formed through oxidative dehydrodimerization of ferulates, producing dehydrodiferulates (henceforth termed diferulates). Such ferulates and diferulates further crosslink plant cell walls by radical coupling cross-reactions during lignification. Although cell wall digestibility can be improved by cell wall metabolic engineering, or post-harvest by various pretreatment processes, a more comprehensive understanding of the role and impact of ferulate crosslinking on polysaccharide hydrolysis would be accelerated by availability of analytical methods that can distinguish the various diferulates released during biomass pretreatments, many of which are isomers. In this report, we present an ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) strategy for comprehensive separation and identification of diferulate isomers. Collision-induced dissociation (CID) mass spectra of [M + H](+) ions distinguished various isomers without requiring derivatization. Characteristic product ions for 8-O-4-, 8-8-non-cyclic, 8-8-cyclic, 8-5-cyclic, 8-5-non-cyclic, and 5-5-linked isomers were identified. All diferulates were identified either as di-acids in extracts of NaOH-hydrolyzed corn stover, or as a diverse group of diferulate mono- and di-amides in extracts of Ammonia Fiber Expansion (AFEX™)-treated corn stover. This approach allows for direct analysis of released diferulates with minimal sample preparation, and can serve as the foundation for high-throughput profiling and correlating pretreatment conditions with biomass digestibility in biorefineries producing biofuels and biochemicals.

Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post-translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF-β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.

Fully automated chip-based nanoelectrospray combined with electron transfer dissociation for high throughput top-down proteomics

Abstract The conventional protocol for protein identification by electrospray ionization mass spectrometry (MS) is based on enzymatic digestion which renders peptides to be analyzed by liquid chromatography-MS and collision-induced dissociation (CID) multistage MS, in the so-called bottom-up approach. Though this method has brought a significant progress to the field, many limitations, among which, the low throughput and impossibility to characterize in detail posttranslational modifications in terms of site(s) and structure, were reported. Therefore, the research is presently focused on the development of procedures for efficient top-down fragmentation of intact protein ions. In this context, we developed here an approach combining fully automated chip-based-nanoelectrospray ionisation (nanoESI), performed on a NanoMate robot, with electron transfer dissociation (ETD) for peptide and top-down protein sequencing and identification. This advanced analytical platform, integrating robotics, microfluidics technology, ETD and alternate ETD/CID, was tested and found ideally suitable for structural investigation of peptides and modified/functionalized peptides as well as for top-down analysis of medium size proteins by tandem MS experiments of significantly increased throughput and sensitivity. The obtained results indicate that NanoMate-ETD and ETD/CID may represent a viable alternative to the current MS strategies, with potential to develop into a method of routine use for high throughput top-down proteomics.

Phosphorylcholine-containing lipid molecular species profiling in biological tissue using a fast HPLC/QqQ-MS method

A fast reversed-phase liquid chromatography-electrospray ionization triple quadrupole mass spectrometry method was developed for the molecular species profiling of glycerophosphocholine (GPC) and sphingomyelin (SM) in total lipid extracts. A two-stage mass spectrometry strategy was adopted to analyze in detail the composition of lipid molecular species. Precursor ion analysis was first conducted to obtain the preliminary composition profile of the phosphorylcholine-containing lipid. The product ion spectra were sequentially acquired for each recorded signal to determine the molecular structure of the lipid. A total of 150 GPCs and 12 SMs were identified in the fetal mouse lung with relative amounts ranging from 13.7% to less than 0.002% (normalizing by the total signal response). A column packed with core-shell particles was used to obtain excellent chromatographic separation with a shorter time demand in a conventional high-performance liquid chromatography system. Considering the compromise between the chromatographic efficiency and the electrospray signal response, the optimization of the mobile phase improves the chromatographic plate number to approximately 40,000 and the detection limits to less than 0.001 mg/L. The applicability of the method was validated through a study of chemically induced early lung maturation. The metabolic alteration in the fetal mouse lung was clearly reflected in the GPC and SM composition with several characteristics of the molecular structure that related to the character of the phospholipid layer upon the epithelial lining of alveoli and the relevant cell function. The results indicated that this analytical strategy is reliable for comprehensive molecular species profiling of GPC and SM and might be extended to the analysis of other phospholipids.

Imaging secondary metabolism of Streptomyces sp. Mg1 during cellular lysis and colony degradation of competing Bacillus subtilis

Soil streptomycetes are saprotrophic bacteria that secrete numerous secondary metabolites and enzymes for extracellular functions. Many streptomycetes produce antibiotics thought to protect vegetative mycelia from competing organisms. Here we report that an organism isolated from soil, Streptomyces sp. Mg1, actively degrades colonies and causes cellular lysis of Bacillus subtilis when the organisms are cultured together. We predicted that the inhibition and degradation of B. subtilis colonies in this competition depends upon a combination of secreted factors, including small molecule metabolites and enzymes. To begin to unravel this complex competitive phenomenon, we use a MALDI imaging mass spectrometry strategy to map the positions of metabolites secreted by both organisms. In this report, we show that Streptomyces sp. Mg1 produces the macrolide antibiotic chalcomycin A, which contributes to inhibition of B. subtilis growth in combination with other, as yet unidentified factors. We suggest that efforts to understand competitive and cooperative interactions between bacterial species benefit from assays that pair living organisms and probe the complexity of metabolic exchanges between them.

Mass spectrometry strategies for mycotoxins analysis in European beers

In this work, an existent solid-phase extraction (SPE) procedure was used to study the occurrence of mycotoxins in different European beers. HPLC-QqQ-MS/MS and ultra high resolution mass analyser have been optimized for the analysis of 18 mycotoxins: the methods were validated according to the EU Commission Decision 2002/657/EC guidelines. In this sense, matrix-matched calibration was performed for each type of beer, obtaining an effective quantification. The recoveries ranged from 63 to 91% and repeatability and reproducibility expressed as relative standard deviation (RSD%) were lower than 17%. On one hand, HPLC-LTQ-Orbitrap® was used for unambiguous identification of target mycotoxins, as well as for screening non-target mycotoxins, such as enniatins, fusaproliferin and deoxynivalenol-3-glucoside. On the other hand, the quantification was carried out using HPLC-QqQ-MS/MS instrument. At the end, Ochratoxin A, fumonisins, HT-2 and T-2 toxins were detected and quantified in European beers. Moreover, pale lager beer showed higher mycotoxin incidence than the other studied beers.

Impact of phosphoproteomics on studies of bacterial physiology

Protein phosphorylation on serine, threonine and tyrosine is recognized as a major tool of signal transduction in bacteria. However, progress in the field has been hampered by the lack of global and site-specific data on bacterial phosphoproteomes. Recent advances in mass spectrometry-based proteomics have encouraged bacteriologists to start using powerful gel-free approaches for global detection of phosphorylated proteins. These studies have generated large data sets of proteins phosphorylated on serine, threonine and tyrosine, with identified phosphorylation sites which represent an excellent starting point for in-depth physiological characterization of kinases and their substrates. The list of phosphorylated proteins inspired a number of physiological studies in which the identity of the phosphorylation site facilitated the elucidation of molecular mechanisms of signaling and regulation. Bacterial phosphoproteomics also provided interesting insights into the evolutionary aspects of protein phosphorylation. The field is rapidly embracing quantitative mass spectrometry strategies, comparing phosphoproteome dynamics in changing conditions and aiming to reconstruct the entire regulatory networks by linking kinases to their physiological substrates.

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative (T(1)), early sporulation (T(2)), and late sporulation (T(3)) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.

Views of the dynamic human spliceosome

The spliceosome is the cellular machinery responsible for removing introns from gene transcripts and ligating together exons to form mature messenger RNAs. It therefore serves as a critical player in regulating human gene expression and proteome complexity. We are working to provide a structural framework to understand the molecular mechanisms of the spliceosome. This task is challenged by size and dynamics of the complex as it forms from over 100 components into several assembly intermediates and functional states. Using an in vitro splicing system in human cell extracts in which we modify the pre‐mRNA substrate upon which the complex is assembled, we have new made advances in capturing the spliceosome at distinct intermediate states relative to spliceosome chemistry. Mass spectrometry analyses of the proteins in the different spliceosome intermediate complexes indicate changes in composition, which we then relate to differences in the structure of the complexes that we observe by electron microscopy. Using a combination of crosslinking, chemical modification and mass spectrometry strategies, we have also identified proteins that change in their association with the RNA substrate as the splicing reaction is catalyzed. Taken together, these observations are building a picture of the molecular rearrangements that occur in the spliceosome during the transition between first and second step chemistry.