A rapid and sensitive method for the identification and quantitation of sub-picomolar amounts of neurohypophysial peptides

A simple, rapid and sensitive method, using isocratic reversed-phase HPLC, is described for the concomitant identification and quantitation of low levels of the various neurohypophysial peptides in biological tissues and fluids. The method requires little or no sample preparation and utilises UV peak detection at 215 nm with a serial signal amplification system to achieve a usable maximum sensitivity of less than 200 fmol of peptide.     

Bioinformatic strategies for unambiguous identification of prostate specific antigen in clinical samples

Prostate specific antigen (PSA), as a widely used clinical biomarker in prostate cancer diagnostics, exists in multiple molecular forms. However, all of these forms might not be recognized in a given sample by the standard immunoassays. Therefore, we have investigated PSA isoforms, separated by size, using mass spectrometric analyses. The objective of these developments was to identify and specify the various forms of PSA. To optimize successful identification of different PSA forms, we have developed a bioinformatic strategy, consisting of high resolution MALDI-MS PMF and sequencing MS/MS data searches. To improve sequence-based identification, the recently introduced Proteios software environment was employed, allowing the combination of multiple database search engines in an automated manner. We could unambiguously identify PSA in clinical samples by all detectable tryptic peptides, which were found to be common in several isoforms.     

Improved strategies for rapid identification of chemically cross-linked peptides using protein interaction reporter technology

Protein interaction reporter (PIR) technology can enable identification of in vivo protein interactions with the use of specialized chemical cross-linkers, liquid chromatography, and high-resolution mass spectrometry. PIR-cross-linkers contain labile bonds that are specifically fragmented under low energy collision or photodissociation conditions in the mass spectrometer source, thus releasing cross-linked peptides. Successful analysis of PIR-cross-linked proteins requires the use of expected mathematical relationships between cross-linked complexes and released peptides after fragmentation of the labile PIR bonds. Presented here is a next-generation software tool, BLinks, for use in the analysis and identification of PIR-cross-linked proteins. BLinks is an advancement beyond our previous efforts by incorporation of chromatographic profiles that must match between cross-linked complexes and released peptides to enable estimation of p-values to help filter true relationships from complex data sets. Additionally, BLinks was used to incorporate Mascot database searching results from subsequent MS/MS analysis of the released peptides to facilitate identification of cross-linked proteins. BLinks was used in the analysis of human serum albumin, and 46 interpeptide relationships were found spanning 30 proximal residues with a 2.2% false discovery rate. BLinks was also used to track peptides involved in multiple, coeluting relationships that make accurate identification of protein interactions difficult. An additional 10 interpeptide relationships were identified despite poor correlation using the profiling tools provided with BLinks. Additionally, BLinks can be used to globally map all interpeptide relationships from the data analysis and customize subsequent analysis to target specific peptides of interest, thus making it a useful tool for both discovery of protein interactions and mapping protein topology.     

Novel and sensitive noncompetitive enzyme immunoassay for peptides

A novel and sensitive noncompetitive enzyme immunoassay for peptides is described. Peptides were biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate and were trapped onto anti-peptide IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated peptides were eluted with HC1 and were reacted with anti-peptide Fab'-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of angiotensin I as a model peptide was 13 fg (10 amol)/tube and 0.8 ng/l of plasma, which was 80 to 480-fold lower than those previously reported by competitive radioimmunoassay and competitive enzyme immunoassay. And other peptides could also be measured more sensitively by the present noncompetitive enzyme immunoassay method than by competitive immunoassays.     

Profiling of endogenous peptides in human synovial fluid by NanoLC-MS: method validation and peptide identification

Synovial fluid potentially contains markers for early diagnosis and disease progression in degenerative joint diseases such as osteoarthritis. Here, a method is described for profiling endogenous peptides in human synovial fluid, using ultrafiltration, solid-phase extraction, nanoscale liquid chromatography, and high-resolution mass spectrometry. Synovial fluid is characterized by its high viscosity, caused by the presence of the lubricant hyaluronic acid. The method proved to be capable of eliminating the high concentrations of hyaluronic acid, which appeared to be necessary to obtain satisfactory analytical performance, that is, within-day relative standard deviations of 5-15%, between-day relative standard deviations of 6-16%, a linear response of R2=0.994, a limit of detection in the femtomole range, and reproducible recoveries of 14-67%. With the developed method, in a synovial fluid sample from an osteoarthritis patient and a healthy control, in total, 501 peptides originating from 40 proteins were identified. Peptide cleavage products from six proteins that have been associated with osteoarthritis in earlier studies (collagen II, proteoglcycan 4, serum amyloid A, tubulin, vimentin, and Matrix Gla) could also be identified with our profiling method. The robustness of the method indicates that it can be applied in systems biology approaches for further studies on degenerative joint disease, eventually leading to a better understanding of the disease and its therapy, as well as the development of novel biomarkers to monitor these processes.     

Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments

Analysis of protein interaction networks and protein complexes using affinity purification and mass spectrometry (AP/MS) is among most commonly used and successful applications of proteomics technologies. One of the foremost challenges of AP/MS data is a large number of false-positive protein interactions present in unfiltered data sets. Here we review computational and informatics strategies for detecting specific protein interaction partners in AP/MS experiments, with a focus on incomplete (as opposite to genome wide) interactome mapping studies. These strategies range from standard statistical approaches, to empirical scoring schemes optimized for a particular type of data, to advanced computational frameworks. The common denominator among these methods is the use of label-free quantitative information such as spectral counts or integrated peptide intensities that can be extracted from AP/MS data. We also discuss related issues such as combining multiple biological or technical replicates, and dealing with data generated using different tagging strategies. Computational approaches for benchmarking of scoring methods are discussed, and the need for generation of reference AP/MS data sets is highlighted. Finally, we discuss the possibility of more extended modeling of experimental AP/MS data, including integration with external information such as protein interaction predictions based on functional genomics data.     

Determination of endogenous peptides in the porcine brain: possible construction of peptidome,a fact database for endogenous peptides

Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.     

C-terminal glyoxylyl peptides for sensitive enzyme-linked immunosorbent assays

A model peptide antigen derived from HCV Coreprotein was modified by a hydrophilic glyoxylamide arm using atartaramide-based linker and used in an enzyme-linkedimmunosorbent assay (ELISA) on hydrazide plates. A comparativestudy with the standard non-covalent adsorption proceduresdemonstrated a large gain in sensitivity for the detection ofantibodies in HCV-positive sera.     

C-terminal glyoxylyl peptides for sensitive enzyme-linked immunosorbent assays

Summary A model peptide antigen derived from HCV Core protein was modified by a hydrophilic glyoxylamide arm using a tartaramide-based linker and used in an enzyme-linked immunosorbent assay (ELISA) on hydrazide plates. A comparative study with the standard non-covalent adsorption procedures demonstrated a large gain in sensitivity for the detection of antibodies in HCV-positive sera.     

Proteomics strategies for protein identification

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.     

A sensitive specific enzymatic-fluorometric assay for homocarnosine

We have developed a sensitive and specific assay for homocarnosine in tissues. Homocarnosine is separated from GABA by ion exchange chromatography. After hydrolysis of homocarnosine with swine kidney carnosinase, the evolved GABA is measured by an enzymatic-fluorometric procedure. As little as 0.1 nmol of tissue homocarnosine can be detected by this procedure. Homoanserine, which would be detected by this assay, can be separated from homocarnosine by thin layer chromatography. No homoanserine could be detected in any tissues examined. There are marked regional variations in levels of homocarnosine in guinea-pig brain that do not correspond to regional differences in GABA levels.     

Investigating endogenous peptides and peptidases using peptidomics

Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters, and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been a successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome--all the peptides in a cell, tissue, or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography--tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation.     

Functional peptide microarrays for specific and sensitive antibody diagnostics

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.