Proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes

Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.     

Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.     

Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.     

Size distribution of native cytosolic proteins of Thermoplasma acidophilum

We used molecular sieve chromatography in combination with LC‐MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and – on the basis of size distribution and bioinformatics analysis – 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition – analyzed by electron microscopy and MALDI‐TOF‐MS, respectively – verified the accuracy of our method.     

Pores for thought: nuclear pore complex proteins

Nuclear pore complexes (NPCs) are enormous macromolecular structures that mediate the active exchange of proteins and RNPs between the nucleus and cytoplasm. Recent work has resulted in a windfall of identified NPC polypeptides, many with unique sequences. Several of the proteins have been shown to be part of extended cytoplasmic and nucleoplasmic NPC filaments. Biochemical, structural and genetic studies on NPC proteins are just beginning to allow an understanding of how they associate into a functional organelle.     

Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion m/z and intensity data in the mascot generic files from LC-ESI-MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC-ESI-MS/MS data from human blood.     

The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes

Mass spectrometry (MS) is a powerful tool for determining the mass of biomolecules with high accuracy and sensitivity. MS performed under so-called “native conditions” (native MS) can be used to determine the mass of biomolecules that associate noncovalently. Here we review the application of native MS to the study of protein−ligand interactions and its emerging role in elucidating the structure of macromolecular assemblies, including soluble and membrane protein complexes. Moreover, we discuss strategies aimed at determining the stoichiometry and topology of subunits by inducing partial dissociation of the holo-complex. We also survey recent developments in "native top-down MS", an approach based on Fourier Transform MS, whereby covalent bonds are broken without disrupting non-covalent interactions. Given recent progress, native MS is anticipated to play an increasingly important role for researchers interested in the structure of macromolecular complexes.     

Proteomics-grade de novo sequencing approach

The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.     

Homology-based functional proteomics by mass spectrometry: application to the Xenopus microtubule-associated proteome

The application of functional proteomics to important model organisms with unsequenced genomes is restricted because of the limited ability to identify proteins by conventional mass spectrometry (MS) methods. Here we applied MS and sequence-similarity database searching strategies to characterize the Xenopus laevis microtubule-associated proteome. We identified over 40 unique, and many novel, microtubule-bound proteins, as well as two macromolecular protein complexes involved in protein translation. This finding was corroborated by electron microscopy showing the presence of ribosomes on spindles assembled from frog egg extracts. Taken together, these results suggest that protein translation occurs on the spindle during meiosis in the Xenopus oocyte. These findings were made possible due to the application of sequence-similarity methods, which extended mass spectrometric protein identification capabilities by 2-fold compared to conventional methods.     

Replication of bacteriophage MS2. X. Phage-specific ribonucleoprotein particles found in MS2-infected Escherichia coli

Phage-specific RNA-protein complexes formed during the MS2 infection process were examined. The fate of ³²P-labeled parental viral RNA was followed to determine what RNA-protein interactions developed early in infection. In order to identify phage-specific ribonucleoprotein complexes at later times in infection, their protein or RNA components were labeled selectively with radioisotopes after suppression of bacterial macromolecular syntheses with Miracil D (Burroughs Wellcome and Co.).     

Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize

Summary A 216 by promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments.     

基于谱图库的蛋白质鉴定策略研究进展

基于质谱的蛋白质组学快速发展,蛋白质质谱数据也呈指数式增长。寻找速度快、准确度高以及重复性好的鉴定方法是该领域的一项重要任务。谱图库搜索策略直接比较实验谱图与谱图库中的真实谱图,充分利用了谱图中的丰度、非常规碎裂模式和其他的一些特征,使得搜索更加快速和准确,成为蛋白质组学的主流鉴定方法之一。文中介绍基于谱图库的蛋白质组质谱数据鉴定策略,并针对其中两个关键步骤——谱图库构建方法和谱图库搜索方法进行深入介绍,探讨了谱图库策略的进展和挑战。     

Protein-RNA cross-linking in the ribosomes of yeast under oxidative stress

Living systems have efficient degradative pathways for dealing with the fact that reactive oxygen species (ROS) derived from cellular metabolism and the environment oxidatively damage proteins and DNA. But aggregation and cross-linking can occur as well, leading to a series of problems including disruption of cellular regulation, mutations, and even cell death. The mechanism(s) by which protein aggregation occurs and the macromolecular species involved are poorly understood. In the study reported here, evidence is provided for a new type of aggregate between proteins and RNA in ribosomes. While studying the effect of oxidative stress induced in the yeast proteome it was noted that ribosomal proteins were widely oxidized. Eighty six percent of the proteins in yeast ribosomes were found to be carbonylated after stressing yeast cell cultures with hydrogen peroxide. Moreover, many of these proteins appeared to be cross-linked based on their coelution patterns during RPC separation. Since they were not in direct contact, it was not clear how this could occur unless it was through the RNA separating them in the ribosome. This was confirmed in a multiple-step process, the first being derivatization of all carbonylated proteins in cell lysates with biotin hydrazide through Schiff base formation. Following reduction of Schiff bases with sodium cyanoborohydride, biotinylated proteins were selected from cell lysates with avidin affinity chromatography. Oxidized proteins thus captured were then selected again using boronate affinity chromatography to capture vicinal diol-containing proteins. This would include proteins cross-linked to an RNA fragment containing a ribose residue with 2',3'-hydroxyl groups. Some glycoproteins would also be selected by this process. LC/MS/MS analyses of tryptic peptides derived from proteins captured by this process along with MASCOT searches resulted in the identification of 37 ribosomal proteins that appear to be cross-linked to RNA. Aggregation of proteins with ribosomal RNA has not been previously reported. The probable impact of this phenomenon cells is to diminish the protein synthesis capacity.     

Comparative Multiplexed Mass Spectrometric Analyses of Endogenously Expressed Yeast Nuclear and Cytoplasmic Exosomes

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the α/β importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.     

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.     

Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance

The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate. The parent ions for MS/MS were randomly and independently sampled from a log-normal population and therefore could be analyzed by ANOVA. Normal distribution analysis confirmed that the parent and fragment ion intensity distributions were sampled over 99.9% of their range that was above the background noise. Arranging the ion intensity data with the identified peptide and protein sequences in structured query language (SQL) permitted the quantification of ion intensity across treatments, proteins and peptides. The intensity of 101,905 fragment ions from 1421 peptide precursors of 583 peptides from 233 proteins separated over 11 sample treatments were computed together in one ANOVA model using the statistical analysis system (SAS) prior to Tukey-Kramer honestly significant difference (HSD) testing. Thus complex mixtures of proteins were identified and quantified with a high degree of confidence using an ion trap without isotopic labels, multivariate analysis or comparing chromatographic retention times.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

MudPIT: multidimensional protein identification technology

Large-scale biology emerged out of the efforts to sequence genomes of important organisms. Based on resources created by whole genome sequencing, large-scale analyses of messenger RNA (mRNA) and protein expression are now possible. With the availability of large amounts of genomic sequence information, a convenient method for the identification and analysis of proteins based on proteolytic digestion into peptides emerged. Processes to fragment peptides using collision-activated dissociation (CAD) in tandem mass spectrometers and computer algorithms to match the tandem mass spectra of peptides to sequences in databases enable rapid identification of amino acid sequences, and hence proteins, present in mixtures. The inherent complexity of the peptide mixtures has necessitated improvements in methodology for mass spectrometry (MS) analysis of peptides.     

Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry

Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA (III)), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13alpha and Cys-125beta. Cys-13alpha was bound to DMA (III) in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125beta was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13alpha > Cys-111alpha > Cys-104alpha and Cys-13alpha > Cys-125beta > Cys-93beta. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.