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PubMed2006

Cleaning of raw peptide MS/MS spectra: improved protein identification following deconvolution of multiply charged peaks, isotope clusters, and removal of background noise.

The dominant ions in MS/MS spectra of peptides, which have been fragmented by low-energy CID, are often b-, y-ions and their derivatives resulting from the cleavage of the peptide bonds. However, MS/MS spectra typically contain many more peaks. These can result not only from isotope variants and multiply charged replicates of the peptide fragmentation products but also from unknown fragmentation pathways, sample-specific or systematic chemical contaminations or from noise generated by the electronic detection system. The presence of this background complicates spectrum interpretation. Besides dramatically prolonged computation time, it can lead to incorrect protein identification, especially in the case of de novo sequencing algorithms. Here, we present an algorithm for detection and transformation of multiply charged peaks into singly charged monoisotopic peaks, removal of heavy isotope replicates, and random noise. A quantitative criterion for the recognition of some noninterpretable spectra has been derived as a byproduct. The approach is based on numerical spectral analysis and signal detection methods. The algorithm has been implemented in a stand-alone computer program called MS Cleaner that can be obtained from the authors upon request.

Adenosine Triphosphatases↗
PubMed2005

Designing a high throughput refolding array using a combination of the GroEL chaperonin and osmolytes.

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL-GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin-substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a 'folding array' to identify superior folding conditions. The stable chaperonin-substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (approximately mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.

Adenosine Triphosphate↗
PubMed2005

Quantitative proteomic analysis of mitochondria from primary neuron cultures treated with amyloid beta peptide.

Increasing evidence supports a role for altered mitochondrial function in the pathogenesis of neuron degeneration in Alzheimer's disease (AD). Although several studies have examined the effect of amyloid beta peptide (Abeta), on activities of individual proteins in primary neuron cultures, there have been no studies of the effects of Abeta on the mitochondrial proteome. Here, we quantitatively measured changes in mitochondrial proteins of primary rat cortical neuron cultures exposed to 25 microM Abeta(25-35) for 16 h using isotope coded affinity tag (ICAT) labeling and 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) which allows simultaneous identification and quantification of cysteine-containing proteins. The analysis of enriched mitochondrial fractions identified 10 proteins including sodium/potassium-transporting ATPase, cofilin, dihydropyrimidinase, pyruvate kinase and voltage dependent anion channel 1 that were statistically significantly (P < 0.05) altered in Abeta-treated cultures. Elevations of proteins associated with energy production suggest that cells undergoing Abeta-mediated apoptosis increase synthesis of proteins essential for ATP production and efflux in an attempt to maintain metabolic function.

Amyloid beta-Peptides↗
PubMed2005

Extraction methods for analysis of citrus leaf proteins by two-dimensional gel electrophoresis.

General procedures for the extraction of Citrus leaf proteins for analysis by two-dimensional electrophoresis (2-DE) were developed through the evaluation and modification of existing methods. Among the methods evaluated, the best results were obtained when Tris-HCl, KCl, and phenol extractions were followed by precipitation with organic solvents to purify and concentrate the samples. The utility of the Tris-HCl extraction method was demonstrated on the leaves of six genetically different Citrus varieties. The methods described are versatile and result in high resolution 2-DE gels of Citrus leaf proteins.

Chemical Fractionation↗
PubMed2005

Multi-domain proteins in the three kingdoms of life: orphan domains and other unassigned regions.

Comparative studies of the proteomes from different organisms have provided valuable information about protein domain distribution in the kingdoms of life. Earlier studies have been limited by the fact that only about 50% of the proteomes could be matched to a domain. Here, we have extended these studies by including less well-defined domain definitions, Pfam-B and clustered domains, MAS, in addition to Pfam-A and SCOP domains. It was found that a significant fraction of these domain families are homologous to Pfam-A or SCOP domains. Further, we show that all regions that do not match a Pfam-A or SCOP domain contain a significantly higher fraction of disordered structure. These unstructured regions may be contained within orphan domains or function as linkers between structured domains. Using several different definitions we have re-estimated the number of multi-domain proteins in different organisms and found that several methods all predict that eukaryotes have approximately 65% multi-domain proteins, while the prokaryotes consist of approximately 40% multi-domain proteins. However, these numbers are strongly dependent on the exact choice of cut-off for domains in unassigned regions. In conclusion, all eukaryotes have similar fractions of multi-domain proteins and disorder, whereas a high fraction of repeating domain is distinguished only in multicellular eukaryotes. This implies a role for repeats in cell-cell contacts while the other two features are important for intracellular functions.

Amino Acid Sequence↗
PubMed2005

Defining putative T cell epitopes from PE and PPE families of proteins of Mycobacterium tuberculosis with vaccine potential.

The identification of T cell epitopes from immune relevant antigens of Mycobacterium tuberculosis is a critical step in the development of a vaccine covering diverse populations. Two multigene families, PE-PGRS and PPE make up about 10% of the M. tuberculosis genome. However, the functions of the proteins coded by these large numbers of genes are unknown. All possible nonameric peptide sequences from PE and PPE proteins were analysed in silico for their ability to bind to 33 alleles of class I HLA. These results reveal that of all PE and PPE proteins, a significant number of these peptides are predicted to be high-affinity HLA binders, irrespective of the length of the protein. The pathogen peptides that could behave as self or partially self-peptides in the host were eliminated using a comparative study with human proteome, thus reducing the number of peptides for analysis. The structural basis for recognition of the nonamers by the respective HLA molecules thus predicted was analyzed by molecular modeling. The structural analysis showed good correlation with the binding prediction. The analysis also led to an understanding of the binding profile of the peptides with respect to different alleles of class I HLA. The predicted epitopes can be tested experimentally for their inclusion in a potential vaccine against tuberculosis that is HLA haplotype-specific.

Alleles↗
PubMed2001

Applied genome research in the field of human vaccines.

Prophylactic vaccination has made an essential contribution to the improvement of human health over the 20th century. However, we still lack efficient vaccines against major human diseases such as malaria or tuberculosis. Today, the design of therapeutic vaccines referred to as 'pharmaccines' is actively investigated in order to treat diseases such as cancer. In that context, novel ways to rationalize and accelerate vaccine discovery are needed. A series of advances in the fields of molecular biology and computer science, have greatly accelerated the rate at which candidate vaccine antigens can be discovered. In this review, we will present and discuss how applied genome research may facilitate antigen discovery and the design of new prophylactic and therapeutic vaccines.

Antigens↗
PubMedDate not supplied

Identification of protein coding genes in genomes with statistical functions based on the circular code.

A new statistical approach using functions based on the circular code classifies correctly more than 93% of bases in protein (coding) genes and non-coding genes of human sequences. Based on this statistical study, a research software called 'Analysis of Coding Genes' (ACG) has been developed for identifying protein genes in the genomes and for determining their frame. Furthermore, the software ACG also allows an evaluation of the length of protein genes, their position in the genome, their relative position between themselves, and the prediction of internal frames in protein genes.

Base Sequence↗
PubMed2002

Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout.

The advent of DNA array technology and proteomics has revolutionised biology by allowing global analysis of cellular events. So far, the benefits from these new techniques have primarily been realised for well-characterised species. These organisms are rarely the most relevant for environmental biology and ecotoxicology. Thus, there is a need to explore new ways to exploit transcriptomics and proteomics for non-model species. In the present study, rainbow trout (Oncorhynchus mykiss) were exposed to a sublethal concentration of waterborne zinc for up to 6 days. The response in gill tissue was investigated by differential screening of a heterologous cDNA array and by protein profiling using Surface Enhanced Laser Desorption/Ionisation (SELDI). The cDNA array, which was a high-density spotted library of cDNA from Fugu rubripes gill, revealed differentially expressed genes related to energy production, protein synthesis, paracellular integrity, and inflammatory response. SELDI analysis yielded seven proteins that were consistently present only in zinc-exposed gills, and four proteins unique to gills from control fish. A further 11 proteins were differentially regulated. Identification of these proteins by bioinformatics proved difficult in spite of detailed information on molecular mass, charge and zinc-binding affinity. It is concluded that these approaches are viable to non-model species although both have clear limitations.

Animals↗
PubMed1999

Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot.

To increase the throughput of protein identification and characterization in proteome studies, we investigated three methods of performing protein digestion in parallel. The first, which we term "one-step digestion-transfer" (OSDT), is based on protein digestion during the transblotting process. It involves the use of membranes containing immobilized trypsin which are intercalated between the gel and a PVDF collecting membrane. During electrotransfer, some digestion of the transferred proteins occurs, although poorly for basic and/or high molecular weight proteins. The second method is based on "in-gel" digestion of all proteins in parallel and termed "parallel in-gel digestion" (PIGD) to denote this fact. The PIGD led to more efficient digestion of basic and high molecular weight proteins (> 40,000) but suffered from a major drawback: loss of resolution for low molecular weight polypeptides (< 60,000) through diffusion during the digestion process. The third method examined was the combination of PIGD and OSDT procedures. This combination, called "double parallel digestion" (DPD), led to greatly improved digestion of high molecular weight and basic proteins without losses of low molecular weight polypeptides. Peptides liberated during transblotting of proteins through the immobilized trypsin membrane were trapped on a PVDF membrane and identified by mass spectrometry in scanning mode.

Bacterial Proteins↗
PubMedDate not supplied

Biomarker discovery in urine by proteomics.

A hypothesis was formed that it would be possible to isolate an adequate amount of protein from a patient, having normal renal function, to identify biological markers of a particular disease state using a variety of proteomics techniques. To support this hypothesis, three samples of urine were collected from a volunteer: first when healthy, later when experiencing acute inflammation due to a pilonidal abcess, and again later still after successful recovery from the condition. The urine from these samples was processed by solid-phase extraction to concentrate and desalt the endogenous proteins and peptides. The proteins and peptides from these urine samples were analyzed in three different experiments: (1) traditional two-dimensional gel electrophoresis followed by proteolysis and mass spectrometric identification of various protein spots, (2) whole mixture proteolysis followed by one-dimensional packed capillary liquid chromatography and tandem mass spectrometry, (3) whole mixture proteolysis followed by two-dimensional capillary liquid chromatography and tandem mass spectrometry. In all three cases, a set of proteins was identified representing putative biomarkers. Each of these proteins was then found to have been previously linked in the scientific literature to inflammation. One acute phase reactant in particular, orosomucoid, was readily observed in all three experiments to dramatically increase in abundance, thereby supporting the hypothesis.

Animals↗
PubMedDate not supplied

Proteome of Haemophilus ducreyi by 2-D SDS-PAGE and mass spectrometry: strain variation, virulence, and carbohydrate expression.

We have analyzed the proteome of several strains of Haemophilus ducreyi by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Over 100 spots were analyzed from the soluble and insoluble protein fractions from the prototype strain 35000HP and 122 distinct proteins were identified. Functions of approximately 80% of the 122 proteins were deduced by identification with close homologues of Haemophilus influenzae. Four additional wild type and three mutant strains were also analyzed that vary in their virulence and/or outer-membrane lipooligosaccharide structures. Overall, the 2-DE gel maps of the wild type and mutant strains were similar to strain 35000HP, suggesting little proteome diversity in relation to carbohydrate expression and/or virulence. An exception was the Kenyan strain 33921 which contained significant differences in its proteome 2-DE map and also synthesizes an unusual LOS with a trisaccharide branch structure. This African strain may represent a prototype of a second clonal group of H. ducreyi.

Carbohydrate Metabolism↗
PubMedDate not supplied

Genome-wide profile of oxidoreductases in viruses, prokaryotes, and eukaryotes.

Enzymes that utilize nicotinamide adenine dinucleotide (NAD) or its 2'-phosphate derivative (NADP) are found throughout the kingdoms of life. These enzymes are fundamental to many biochemical pathways, including central intermediary metabolism and mechanisms for cell survival and defense. The complete genomes of 25 organisms representing bacteria, protists, fungi, plants, and animals, and 811 viruses, were mined to identify and classify NAD(P)-dependent enzymes. An average of 3.4% of the proteins in these genomes was categorized as NAD(P)-utilizing proteins, with highest prevalence in the medium-chain oxidoreductase and short-chain oxidoreductase families. In general, the distribution of these enzymes by oxidoreductase family was correlated to the number of different catalytic mechanisms in each family. Organisms with smaller genomes encoded a larger proportion of NAD(P)-dependent enzymes in their proteome (approximately 6%) as compared to the larger genomes of eukaryotes (approximately 3%). Among viruses, those with large, double-strand DNA genomes were shown to encode oxidoreductases. Gram-positive and gram-negative bacteria showed some differences in the distribution of NAD(P)-dependent proteins. Several organisms such as M. tuberculosis, P. falciparum, and A. thaliana showed unique distributions of oxidoreductases corresponding to some phenotypic features.

Animals↗
PubMed2006

Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis.

Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10,000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.

Animals↗
PubMed2006

Protein bipartivity and essentiality in the yeast protein-protein interaction network.

Protein-protein interaction networks (PINs) are structured by means of a few highly connected proteins linked to a large number of less-connected ones. Essential proteins have been found to be more abundant among these highly connected proteins. Here we demonstrate that the likelihood that removal of a protein in a PIN will prove lethal to yeast correlates with the lack of bipartivity of the protein. A protein is bipartite if it can be partitioned in such a way that there are two groups of proteins with intergroup, but not intragroup, interactions. The abundance of essential proteins found among the least bipartite proteins clearly exceeds that found among the most connected ones. For instance, among the top 50 proteins ranked by their lack of bipartivity 62% are essential proteins. However, this percentage is only 38% for proteins ranked according to their number of interactions. Protein bipartivity also surpasses another 5 measures of protein centrality in yeast PIN in identifying essential proteins and doubles the number of essential proteins selected at random. We propose a possible mechanism for the evolution of essential proteins in yeast PIN based on the duplication-divergence scheme. We conclude that a replica protein evolving from a nonbipartite target will also be nonbipartite with high probability. Consequently, these new replicas evolving from nonbipartite (essential) targets will with high probability be essential.

Evolution, Molecular↗
PubMed2006

Multi-Q: a fully automated tool for multiplexed protein quantitation.

The iTRAQ labeling method combined with shotgun proteomic techniques represents a new dimension in multiplexed quantitation for relative protein expression measurement in different cell states. To expedite the analysis of vast amounts of spectral data, we present a fully automated software package, called Multi-Q, for multiplexed iTRAQ-based quantitation in protein profiling. Multi-Q is designed as a generic platform that can accommodate various input data formats from search engines and mass spectrometer manufacturers. To calculate peptide ratios, the software automatically processes iTRAQ's signature peaks, including peak detection, background subtraction, isotope correction, and normalization to remove systematic errors. Furthermore, Multi-Q allows users to define their own data-filtering thresholds based on semiempirical values or statistical models so that the computed results of fold changes in peptide ratios are statistically significant. This feature facilitates the use of Multi-Q with various instrument types with different dynamic ranges, which is an important aspect of iTRAQ analysis. The performance of Multi-Q is evaluated with a mixture of 10 standard proteins and human Jurkat T cells. The results are consistent with expected protein ratios and thus demonstrate the high accuracy, full automation, and high-throughput capability of Multi-Q as a large-scale quantitation proteomics tool. These features allow rapid interpretation of output from large proteomic datasets without the need for manual validation. Executable Multi-Q files are available on Windows platform at http://ms.iis.sinica.edu.tw/Multi-Q/.

Chromatography, Liquid↗