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PubMed2006

Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers.

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.

Amino Acid Sequence↗
PubMed2005

Screening for the expression of soluble recombinant protein in Escherichia coli.

Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines. Escherichia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility, and toxicity in this environment, resulting in differing rates of protein degradation, formation into insoluble inclusion bodies, and cell death, thus affecting the amount of soluble protein that can be obtained from E. coli grown in culture. To take full advantage of a variety of strategies developed to improve the expression of soluble protein in E. coli, an easy, rapid means to test many growth parameters is necessary. This chapter describes a dot-blot expression screen to test the effects of growth and induction parameters on the yield of soluble protein. The expression screen is used to detect hexahistidine-tagged proteins expressed in E. coli; however, it is adaptable for the detection of other affinity tags or fusion partners that have suitable antibodies available. In this example, induction time and temperature are tested; however, it can be used to test additional parameters, such as affinity tag type and placement, E. coli host type, and growth medium formulations.

Animals↗
PubMed2003

Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes.

A series of protein expression vectors with dual-affinity tags has been developed. With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations. Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors. These vectors allow evaluation of different positions and orders of two different tags. To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex. First, an appropriate position of the tags was selected through small-scale purification. Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification. These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.

Acetyltransferases↗
PubMed2004

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana.

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.

Antioxidants↗
PubMed2004

Activity profiling of papain-like cysteine proteases in plants.

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

Amino Acid Sequence↗
PubMed2001

Evaluation of an antibody-based genotype classification of the platelet fibrinogen receptor (GPIIb/IIIa).

Platelet membrane glycoprotein (GP) IIb/IIIa is the central molecule in platelet adhesion and aggregation by high-affinity binding of fibrinogen. Polymorphism of the beta chain of the receptor, especially the GPIIIa-proline33 allele [HPA-1b, Zwb, PI(A2)], has been suggested to be associated with a variety of vascular diseases, such as coronary stenosis, myocardial infarction, cerebral ischemia, or venous thrombosis. Using clinical chemistry standards, we evaluate a flow cytometric whole-blood, antibody-based method to determine the genotype [PI(A1A1), PI(A1A2), PI(A2A)] versus polymerase chain reaction (PCR)-based DNA restriction fragment length analysis in 220 individuals. Both homozygous and heterozygous genotypes differ in the expression of binding sites for the monoclonal antibody, SZ21. Agreement between the two methods was achieved in 187 cases, which reflects a test validity of 85%, a sensitivity of 83.6%, and a specificity of 85.4%. We conclude that flow cytometry is reliable for classifying the PI(AX) genotype. The performance characteristics are easy, fast, and cheap (genomics by proteomics). These features make it suitable for screening patients and broad populations for the future risk of cardiovascular ischemic events.

Blood Platelets↗
PubMed2006

[Aptamers in clinical diagnostics].

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro from combinatorial libraries in a process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Aptamers play a role of artificial nucleic acid ligands that can recognize and bind to various organic or inorganic target molecules with high specificity and affinity. They can discriminate even between closely related targets and can be easily chemically modified for radioactive, fluorescent and enzymatic labeling or biostability improvement. Aptamers can thus be considered as universal receptors that rival antibodies in diagnostics as a tool of molecular recognition. To date aptamers have been successively used instead of monoclonal antibodies in flow cytometry, immunochemical sandwich assays and in vivo imaging as well to detect wide range of small or large biomolecules.

Aptamers, Nucleotide↗
PubMed2006

Peptide length-based prediction of peptide-MHC class II binding.

MOTIVATION: Algorithms for predicting peptide-MHC class II binding are typically similar, if not identical, to methods for predicting peptide-MHC class I binding despite known differences between the two scenarios. We investigate whether representing one of these differences, the greater range of peptide lengths binding MHC class II, improves the performance of these algorithms. RESULTS: A non-linear relationship between peptide length and peptide-MHC class II binding affinity was identified in the data available for several MHC class II alleles. Peptide length was incorporated into existing prediction algorithms using one of several modifications: using regression to pre-process the data, using peptide length as an additional variable within the algorithm, or representing register shifting in longer peptides. For several datasets and at least two algorithms these modifications consistently improved prediction accuracy. AVAILABILITY: http://malthus.micro.med.umich.edu/Bioinformatics

Algorithms↗
PubMed2004

Sample preparation of gel electrophoretically separated protein binding partners for analysis by mass spectrometry.

The identification and characterization of binding partners from protein complexes is increasingly undertaken by mass spectrometry because of its high sensitivity and expedient elucidation of protein structure by accurate mass measurement. A variety of affinity purification methods including immunoprecipitation and glutathione-S-transferase (GST) pull-downs are commonly employed for the isolation of protein complexes and coupled to gel electrophoresis for further separation and basic information with regard to their constituents. For the successful analysis of gel-separated proteins by mass spectrometry, additional sample preparation steps involving sample clean-up, proteolysis, and peptide recovery are essential. This chapter describes the important procedure of in-gel digestion with particular emphasis on maximum peptide recovery and compatibility for subsequent mass spectrometric analysis.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.

A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

Botulinum Toxins↗
PubMed2026

Olive leaf protein hydrolysates yield gastro-resistant peptides with antioxidant and anti-inflammatory potential: peptidomics, in vitro validation and molecular docking analyses.

Olive (Olea europaea L.) leaves are an abundant olive-oil by-product and a promising feedstock for sustainable valorisation. An olive leaf protein isolate (OLPI) from olive-leaf powder (OLP) was enzymatically hydrolysed to yield seven hydrolysates (OLPHs). All showed notable antioxidant activity as whole hydrolysate matrices (EC₅₀ = 0.11-0.28 mg mL-1); likely reflecting the combined contribution of released peptides and co-extracted phenolic compounds; the 15-min Alcalase product (OLPH15A) showed high activity with the shortest processing time. Its INFOGEST digest (dOLPH15A) attenuated LPS-induced inflammation in Caco-2 cells, down-regulating pro-inflammatory and up-regulating anti-inflammatory genes. Peptidomics identified 7037 peptides in OLPH15A and 534 in dOLPH15A, from which twenty gastro-resistant sequences were prioritised for in silico analysis. Multi-tool prediction and docking highlighted four peptides, GAAGGIGQPL, QSAYPGTGPL, GGGAGGGDGGIL and LDAQFPGVN, with favourable predicted affinity for the TLR4/MD2 complex, suggesting that they may contribute to the observed immunomodulatory response. These findings position olive leaves as a viable source of protein hydrolysate-based ingredients with antioxidant and anti-inflammatory potential, advancing the valorisation of olive-oil by-products.

Olea↗
PubMed2003

Separation and detection methods for covalent drug-protein adducts.

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.

Chromatography, High Pressure Liquid↗
PubMed2004

Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture.

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.

Amino Acid Sequence↗
PubMed2004

Array assessment of phage-displayed peptide mimics of Human Immunodeficiency Virus type 1 gp41 immunodominant epitope: binding to antibodies of infected individuals.

An array of phage-displayed mimetic peptides representing 18 sequences from the HIV-1 gp41 immunodominant epitope was fabricated on a nitrocellulose membrane. It used to compare the antibody-binding affinity of the peptide and to monitor the immune response of four patients prior to and after the initiation of Highly Active Anti-Retroviral Therapy in parallel assays with several probes using near-infrared fluorescence detection. The proposed multiplexed approach is highly sensitive, consumes less sample and can be used to analyze the immune repertoires of virus-infected individuals.

Amino Acid Sequence↗
PubMed2000

Convergent evolution with combinatorial peptides.

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.

Amino Acid Sequence↗
PubMed2005

Average peptide score: a useful parameter for identification of proteins derived from database searches of liquid chromatography/tandem mass spectrometry data.

The quantity and variable quality of data that can be generated from liquid chromatography (LC)/mass spectrometry (MS)-based proteomics analyses creates many challenges in interpreting the spectra in terms of the actual proteins in a complex sample. In spite of improvements in algorithms that match putative peptide sequences to MS/MS spectra, the assembly of these lists of possible or probable peptides into a 'correct' set of proteins is still problematic. We have observed a trend in a simple relationship, derived from standard database search outputs, which can be useful in assessing the quality of a MS/MS-based protein identification. Specifically, the ratio of the protein score and number of non-redundant peptides, or average peptide score (APS), can facilitate initial filtering of database search results in addition to providing a useful measure of confidence for the proteins identified. This parameter has been applied to results from the analysis of multi-protein complexes derived from pull-down experiments analyzed using a two-dimensional LC/MS/MS workflow. In particular, the complex list of protein identifications derived from a drug affinity pull-down with immobilized ampicillin and an E. coli lysate was greatly simplified by applying the APS as a filter, allowing for facile identification of the penicillin-binding proteins known to interact with ampicillin. Furthermore, an APS threshold can be used for any data sets derived from electrospray ionization (ESI)- or matrix-assisted laser desorption/ionization (MALDI)-MS/MS experiments and is also not specific to any database search program.

Chromatography, Liquid↗
PubMed2005

Comparative glycoproteomics of N-linked complex-type glycoforms containing sialic acid in human serum.

This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.

Amino Acid Sequence↗
PubMed2005

Protein-detecting microarrays: current accomplishments and requirements.

The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry will provide simple production protocols and specific orientations of capture agents on the microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery.

Animals↗