Building consensus spectral libraries for peptide identification in proteomics

Spectral searching has drawn increasing interest as an alternative to sequence-database searching in proteomics. We developed and validated an open-source software toolkit, SpectraST, to enable proteomics researchers to build spectral libraries and to integrate this promising approach in their data-analysis pipeline. It allows individual researchers to condense raw data into spectral libraries, summarizing information about observed proteomes into a concise and retrievable format for future data analyses.     

Combinatorial methods for discovery of peptide ligands which bind to antibody-like molecules

In the 1980s, new methods of parallel peptide synthesis were used to make large libraries of peptides, which were then screened for binding to Bence-Jones dimers. Subsequent X-ray crystallography of the Bence-Jones proteins, which had been infiltrated with peptide ligand, was used to determine the structural correlate of the binding data. The mode of binding was found to be not predictable and insight was gained into the forces determining how the so-called mimotopes interacted with binding sites.     

Combinatorial libraries: a tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity relationship studies

In the race for supremacy, microbes are sprinting ahead. This warning by the World Health Organization clearly demonstrates that the spread of antibiotic-resistant bacteria leads to a global health problem and that antibiotics never seen before by bacteria are urgently needed. Antimicrobial peptides represent such a source for novel antibiotics due to their rapid lytic activity (within minutes) through disruption of cell membranes. However, due to the similarities between bacterial, fungal, and mammalian plasma cell membranes, a large number of antimicrobial peptides have low lytic specificities and exhibit a broad activity spectrum and/or significant toxic effect toward mammalian cells. Mutation strategies have allowed the development of analogues of existing antimicrobial peptides having greater lytic specificities, although such methods are lengthy and would be more efficient if the molecular mechanisms of action of antimicrobial peptides were clearly elucidated. Synthetic combinatorial library approaches have brought a new dimension to the design of novel biologically active compounds. Thus, a set of peptide analogues were generated based on the screening of a library built around an existing lytic peptide, and on a deconvolution strategy directed toward activity specificity. These peptide analogues also served as model systems to further study the effect of biomembrane mimetic systems on the peptides structural behavior relevant to their biological activities.     

Multidimensional peptide separations in proteomics

Multidimensional peptide separation will play an increasingly important role in the drive to identify and quantitate the proteome. By increasing the peak and load capacity, multidimensional approaches increase the number and dynamic range of peptides that can be analyzed in a complex biological organism. Separation methods using different physical properties of peptides have been combined with varying degrees of success. The ultimate goal is a rapid separation strategy that can be coupled with analytical methods, such as mass spectrometry, to provide comprehensive monitoring of the changing concentration, interactions, and structures of proteins in the proteome.     

Building and searching tandem mass (MS/MS) spectral libraries for peptide identification in proteomics

Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. In spectral library searching, a spectral library is first meticulously compiled from a large collection of previously observed peptide MS/MS spectra that are conclusively assigned to their corresponding amino acid sequence. An unknown spectrum is then identified by comparing it to all the candidates in the spectral library for the most similar match. This review discusses the basic principles of spectral library building and searching, describes its advantages and limitations, and provides a primer for researchers interested in adopting this new approach in their data analysis. It will also discuss the future outlook on the evolution and utility of spectral libraries in the field of proteomics.     

Modern methods to produce natural-product libraries

Natural sources offer a wealth of chemically diverse compounds that have been evolutionary preselected to modulate biochemical pathways. Several industrial and academic groups are accessing this source using advanced technology platforms. Methods have been reported to generate large and diverse natural-product libraries optimised for high-throughput screening and for a fast discovery process. In addition to prefractionated and pure natural-product libraries, parallel synthesis gives access to synthetic, semi-synthetic and natural-product-like libraries. Natural-product chemistry and organic synthesis are powerful tools for optimising natural leads and for generating new diversity from natural scaffolds. The amalgamation of both may be expected to become an important strategy in future drug design.     

The audacity of proteomics: a chance to overcome current challenges in schizophrenia research

The evidence on the mechanisms underlying schizophrenia seems to support a series of divergent possibilities that may play a contributory role in the disease pathogenesis. Hypotheses regarding disease mechanisms range from structural and neurodevelopmental abnormalities to neurotransmitter deficits and genetic predisposition. Nevertheless, little progress has been made in the field of schizophrenia with respect to the key pathophysiological processes, and this is a fundamental barrier to identifying or predicting the therapeutic value of novel drug targets. All multi-omics approaches, including proteomics, would be perfectly suited to such a complex disease as a means of tackling the widening chasm between the aforementioned hypotheses. Proteomic studies have been performed in a variety of tissues and biological fluids, from post-mortem CNS tissue to cerebrospinal fluid and blood, and in recently generated mutant animal models with schizophrenia-like phenotypes. In this review, we present an overview of several proteomic studies in the field of schizophrenia research and discuss how proteomics could assist with identifying schizophrenia markers more efficiently, covering both clinical and basic research.     

Combinatorial methods in molecular imprinting

Molecular imprinting is a general method for synthesizing robust, network polymers with highly specific binding sites for small molecules. Recently, combinatorial and computational approaches have been employed to select an optimal molecularly imprinted polymer (MIP) formulation for a targeted analyte. The use of MIPs in the combinatorial field, specifically their use for screening libraries of small molecules, has also been developed.     

Gel-based methods in redox proteomics

Background

The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification.

Scope of the review

This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated.

Major conclusions

A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein.

General significance

By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Complementary methods to assist subcellular fractionation in organellar proteomics

Organellar proteomics aims to describe the full complement of proteins of subcellular structures and organelles. When compared with whole-cell or whole-tissue proteomes, the more focused results from subcellular proteomic studies have yielded relatively simpler datasets from which biologically relevant information can be more easily extracted. In every proteomic study, the quality and purity of the biological sample to be investigated is of the utmost importance for a successful analysis. In organellar proteomics, one of the most crucial steps in sample preparation is the initial subcellular fractionation procedure by which the enriched preparation of the sought-after organelle is obtained. In nearly all available organellar proteomic studies, the method of choice relies on one or several rounds of density-based gradient centrifugation. Although this method has been recognized for decades as yielding relatively pure preparations of organelles, recent technological advances in protein separation and identification can now reveal even minute amounts of contamination, which in turn can greatly complicate data interpretation. The scope of this review focuses on recently published innovative complementary or alternative methods to perform subcellular fractionation, which can further refine the way in which sample preparation is accomplished in organellar proteomics.     

Peptide libraries: at the crossroads of proteomics and bioinformatics

Peptide libraries offer a valuable means for providing functional information regarding protein-modifying enzymes and protein interaction domains. Library approaches have become increasingly useful as high-throughput strategies for the analysis of large numbers of new proteins identified as a result of genome-sequencing efforts. Recent developments in the field have produced faster methods with broadened applicability. Crucially, new computational and biochemical tools have emerged that facilitate identification of interaction partners and substrates for proteins on the basis of their peptide selectivity profiles. Such combinations of proteomics-scale experimental approaches with bioinformatics tools hold great promise for the elucidation of protein interaction networks and signal transduction pathways in living cells.     

Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage.

An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors. This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd. A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed. The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1. Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described. Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames. Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.     

Combinatorial peptide ligand libraries for the analysis of low-expression proteins: Validation for normal urine and definition of a first protein MAP

In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorial peptide ligand library (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are (i) definition of the urine sample requirements, (ii) optimization of the urine/ligand ratio, (iii) essay conditions, (iv) en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter and intraessay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC-MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly developed studies on human diseases.     

Histidine-rich peptide selection and quantification in targeted proteomics

Agarose based immobilized copper (II) affinity chromatography (Cu(II)-IMAC) in tandem with reversed-phase chromatography was applied to a yeast protein extract. Histidine-rich peptides were selected and, in the process, samples were substantially simplified prior to mass spectral analysis. Samples of proteins from the yeast extract at fermentation time periods of 2.5 and 10 h were compared quantitatively used the GIST protocol. Acylation of the N-terminus of tryptic peptides with N-acetoxysuccinamide was used to globally label and quantify relative protein concentration changes. Together with N-terminal acylation, an imidazole elution procedure allowed histidine-rich peptides to be preferentially selected by Cu(II)-IMAC. An inverse labeling strategy was applied to increase reliability in determinations of up- and down-regulation. It was found that the concentration of some histidine-rich proteins changed in excess of 4-fold during fermentation. These proteins covered a wide range of molecular weight and pI values.     

From peptide libraries to optimized nonpeptide ligands in the search for S-farnesyltransferase inhibitors.

A complete 331,776-member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S-farnesyltransferase activity in vitro. One of the non-natural peptide and noncysteine-containing leads Nip-Trp-Phe-His (Nip=p-nitrophenyl-L-alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200-fold potency compared with the original lead. The final compound was converted to the C-terminal ethyl ester: p-F-C6H4-CO(CH2)2-CO-Bta-D-Phepsi[CH2NH]His-OEt (Bta = benzothienyl-L-alanine) and shown to behave as a prodrug which was hydrolyzed back to the C-terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.     

Biodistribution of filamentous phage peptide libraries in mice

In vivo phage display is a new approach to acquire peptide molecules that bind stably to a given target. Phage peptide display libraries have been selected in mice and humans and numerous vasculature-targeting peptides have been reported. However, in vivo phage display has not typically produced molecules that extravasate to target specific organ or tumor antigens. Phage selections in animals have been performed for very short times without optimization for biodistribution or clearance rates to a particular organ. It is hypothesized that peptides that home to a desired antigen/organ can be obtained from in vivo phage experiments by optimization of incubation times, phage extraction and propagation procedures. To accomplish this goal, one must first gain a better understanding of the in vivo biodistribution and rate of clearance of engineered phage peptide display libraries. While the fate of wild type phage in rodents has been reported, the in vivo biodistribution of the commonly used engineered fd-tet M13 phage peptide display libraries (such as in the fUSE5 vector system) have not been well established. Here we report the biodistribution and clearance properties of fd-tet fifteen amino acid random peptide display libraries in fUSE5 phage in three common mouse models employed for drug discovery - CF-1, nude, and SCID mice.     

Combinatorial peptide microarrays for the rapid determination of kinase specificity

We report a rapid method for profiling of kinases using a strategy that couples the merits of combinatorics (in rapid diversity generation) with the throughput attainable using microarrays (in parallel screening). Alanine-scanning, deletion and positional-scanning peptide libraries of a kinase substrate were synthesized and site-specifically arrayed onto glass slides. The phosphorylation pattern of target sequences detected using fluorescently-labeled antiphosphoamino acid antibodies revealed the substrate preference of the kinase through its activity profile.