Mass Spectrometry-based Peptidomics Research Articles

Analysis of the Yeast Peptidome and Comparison with the Human Peptidome.

Peptides function as signaling molecules in species as diverse as humans and yeast. Mass spectrometry-based peptidomics techniques provide a relatively unbiased method to assess the peptidome of biological samples. In the present study, we used a quantitative peptidomic technique to characterize the peptidome of the yeast Saccharomyces cerevisiae and compare it to the peptidomes of mammalian cell lines and tissues. Altogether, 297 yeast peptides derived from 75 proteins were identified. The yeast peptides are similar to those of the human peptidome in average size and amino acid composition. Inhibition of proteasome activity with either bortezomib or epoxomicin led to decreased levels of some yeast peptides, suggesting that these peptides are generated by the proteasome. Approximately 30% of the yeast peptides correspond to the N- or C-terminus of the protein; the human peptidome is also highly represented in N- or C-terminal protein fragments. Most yeast and humans peptides are derived from a subset of abundant proteins, many with functions involving cellular metabolism or protein synthesis and folding. Of the 75 yeast proteins that give rise to peptides, 24 have orthologs that give rise to human and/or mouse peptides and for some, the same region of the proteins are found in the human, mouse, and yeast peptidomes. Taken together, these results support the hypothesis that intracellular peptides may have specific and conserved biological functions.

Protection of Brain Injury by Amniotic Mesenchymal Stromal Cell-Secreted Metabolites.

To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. Prospective experimental study. Laboratory research. C57Bl/6 mice. Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and containing neither proteic nor ribonucleic molecules was protective. This study indicates the profiling of protective factors that could be useful in cell-free therapeutic approaches for acute brain injury.

Analytic framework for peptidomics applied to large-scale neuropeptide identification.

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.

Ethnobotanical survey of Rinorea dentata (Violaceae) used in South-Western Nigerian ethnomedicine and detection of cyclotides

Ethnopharmacological relevancePeople living in the tropical rain forest of South-Western Nigeria use Rinorea dentata (P. Beauv.) Kuntze (Violaceae) in ethno-veterinary medicine to facilitate parturition. There are no evidence-based pharmacological investigations for the uterotonic activity of this plant. Aims of study(i) Collection of data about the ethnopharmacological uses of R. dentata and evaluation of its uses and applications in health care; (ii) determining potential uterotonic effects in vitro, and (iii) chemical characterization of R. dentata, which is a member of the Violaceae family known to express circular cystine-knot peptides, called cyclotides. Materials and methodsThe ethnopharmacological use of R. dentata in settlement camps within the area J4 of Omo forest has been investigated by semi-structured questionnaires and open interviews. Use index analysis has been performed by seven quantitative statistical models. Respondents’ claim on the beneficial ethno-veterinary application of the plant to aid parturition has been investigated in vitro by myometrial contractility organ bath assays. The bioactive plant extract was screened by chemical derivatization and mass spectrometry-based peptidomics using reversed-phase HPLC fractionation and MALDI-TOF/TOF analysis. ResultsBased on the survey analysis, medicinal preparations of R. dentata have been used for anti-microbial and anti-malaria purpose in humans, and for aiding parturition in farm animals. The latter application was mentioned by one out of six respondents who claimed to use this plant for any medicinal purpose. The plant extract exhibited a weak uterotonic effect using organ bath studies. The plant contains cyclotides and the peptide riden A has been identified by de novo amino acid sequencing using mass spectrometry. ConclusionFew dwellers around the settlement camps of the tropical forest of Omo (Nigeria) use R. dentata for various health problems in traditional veterinary and human medicine. The weak uterotonic effect of the cyclotide-rich extract is in agreement with the low use value index obtained for this plant. Cyclotides have been reported in the genus Rinorea confirming the ubiquitous expression of these stable bioactive plant peptides within the family of Violaceae.

Unravelling peptidomes by in silico mining

AbstractPeptides of great number and diversity occur in all domains of life and exhibit a range of pharmaceutically relevant bioactivities. The complexity of biological samples including human cells or tissues, plant extracts or animal venom cocktails, often impedes the discovery of novel bioactive peptides using mass spectrometrybased peptidomics analysis. An increasing number of publicly available genome and transcriptome datasets, together with refined bioinformatics analysis, allows for rapid identification of novel peptides which may have been previously unrecognized. Moreover, a combination of information extracted from

Peptidomic analysis of healthy and subclinically mastitic bovine milk

A variety of proteases release hundreds of endogenous peptide fragments from intact bovine milk proteins. Mass spectrometry-based peptidomics allows for high throughput sequence assignment of a large number of these peptides. Mastitis is known to result in increased protease activity in the mammary gland. Therefore, we hypothesized that subclinically mastitic milks would contain higher concentrations of released peptides. In this work, milks were sampled from three cows and, for each, one healthy and one subclinically mastitic teat were sampled for milk. Peptides were analyzed by nano-liquid chromatography quadrupole time of flight tandem mass spectrometry and identified with database searching. In total, 682 peptides were identified. The total number of released peptides increased 146% from healthy to subclinically mastitic milks (p < 0.05), and the total abundance of released peptides also increased significantly (p < 0.05). Bioinformatic analysis of enzyme cleavage revealed increases in activity of cathepsin D and elastase (p < 0.05) with subclinical mastitis.

Mass spectrometry-based serum and plasma peptidome profiling for prediction of treatment outcome in patients with solid malignancies.

Treatment selection tools are needed to enhance the efficacy of targeted treatment in patients with solid malignancies. Providing a readout of aberrant signaling pathways and proteolytic events, mass spectrometry-based (MS-based) peptidomics enables identification of predictive biomarkers, whereas the serum or plasma peptidome may provide easily accessible signatures associated with response to treatment. In this systematic review, we evaluate MS-based peptide profiling in blood for prompt clinical implementation. PubMed and Embase were searched for studies using a syntax based on the following hierarchy: (a) blood-based matrix-assisted or surface-enhanced laser desorption/ionization time-of-flight MS peptide profiling (b) in patients with solid malignancies (c) prior to initiation of any treatment modality, (d) with availability of outcome data. Thirty-eight studies were eligible for review; the majority were performed in patients with non-small cell lung cancer (NSCLC). Median classification prediction accuracy was 80% (range: 66%-93%) in 11 models from 14 studies reporting an MS-based classification model. A pooled analysis of 9 NSCLC studies revealed clinically significant median progression-free survival in patients classified as "poor outcome" and "good outcome" of 2.0 ± 1.06 months and 4.6 ± 1.60 months, respectively; median overall survival was also clinically significant at 4.01 ± 1.60 months and 10.52 ± 3.49 months, respectively. Pretreatment MS-based serum and plasma peptidomics have shown promising results for prediction of treatment outcome in patients with solid tumors. Limited sample sizes and absence of signature validation in many studies have prohibited clinical implementation thus far. Our pooled analysis and recent results from the PROSE study indicate that this profiling approach enables treatment selection, but additional prospective studies are warranted.

Using Mass Spectrometry-Based Peptidomics to understand the Brain and Disorders such as Parkinson’s Disease and Schizophrenia

The numerous efforts invested in the identification of biomarkers for neurodegenerative and neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, are justified because these disorders affect several million people worldwide. Although genetic implications and the role of the environment have been shown in the progression of those disorders, together with anatomical and neurochemical characteristics, an integrated view of the biochemical pathways involved in the pathophysiology of these disorders is still being unraveled. The use of proteomic methodologies, molecular mechanisms and potential biomarker candidates for the prognosis, diagnosis and treatment of brain disorders has been discussed. Similar methodologies can be applied for the large-scale identification of peptides to characterize the brain peptidome with the aim of closing the knowledge gaps that remain. Brain cells contain a large number of peptides that play pivotal roles in cell communication. Peptidome studies have recently identified more than 800 peptides in mouse brain extracts, with half of them derived from secretory pathways. For example, several of these peptides were identified as bioactive neuropeptides that activate G-coupled receptors. In addition, intracellular peptides derived from nuclear, cytosolic and mitochondrial proteins have been identified, including the hemopressins, which act with high selectivity for the cannabinoid receptor type 1. Considering the importance of peptides in cell signaling, the present review intends to discuss the recent findings of the peptidome field, focusing on Parkinson's disease and schizophrenia. New approaches to evaluate intracellular peptide signaling at the protein-protein interaction level and the future perspectives of peptides as intracellular modulators of signal transduction are explored.

Molecular characterization ofSaccharomyces cerevisiaeα-pheromone by mass spectrometry-based peptidomics

Using modern peptide analytical MS technology ('Peptidomics'), it is possible to analyze yeast α-pheromone both qualitatively and semi-quantitatively directly from conditioned cell culture media. MS/MS analysis shows both forms of α-pheromone (MFα and MFα') detectable and identifiable straight from WT supernatants. In addition to the mature intact α-pheromones, also post-translationally modified α-pheromone peptides and fragments thereof are found to be present in the culture medium. This molecular analytical technique is complementary to the recently described quantitation method by Rogers etal. (2012, FEMS Yeast Res. 12:668) based on ELISA.

Fatty Acid and Peptide Profiles in Plasma Membrane and Membrane Rafts of PUFA Supplemented RAW264.7 Macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

A Comprehensive Peptidome Profiling Technology for the Identification of Early Detection Biomarkers for Lung Adenocarcinoma

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000–5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273–283, FIBA 5–16, and LBN 306–313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.

Peptidomics of the Prolyl Peptidases

The prolyl peptidases are a family of enzymes characterized by a biochemical preference for cleaving proline-containing peptides. The members of this enzyme family include prolyl endopeptidase, prolyl endopeptidase-like, dipeptidyl peptidase 4 (DPP4), DPP7, DPP8, DPP9, and fibroblast activation protein. DPP4 is the best studied member of the family, due to its role in physiological glucose tolerance, exerted through the regulation of the insulinotropic peptide glucagon-like peptide-1. While other members of the prolyl peptidase family have also been implicated in various (patho)physiological processes, the underlying peptides and pathways regulated by these enzymes are less clear. The identification of endogenous substrates of the prolyl peptidases is an important step in elucidating the molecular mechanisms of these enzymes. Here, we highlight the utility of liquid chromatography-mass spectrometry-based peptidomics to enable the discovery of endogenous prolyl peptidase substrates directly from tissues, and demonstrate the utility of this information in understanding the biochemical and physiological functions of the prolyl peptidases.

Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor

High-resolution mass spectrometry-based peptidomics has been used to characterize several components in electro-stimulated skin secretions of the endemic Mexican frog Pachymedusa dacnicolor. Peptide mass screening performed in an Orbitrap-XL mass spectrometer showed that P. dacnicolor skin secretions possess 194 different components with molecular masses ranging mainly from 500 to 6,000 Da. Dozens of molecules were partially sequenced including two novel protease inhibitors. Additionally, one posttranslationally modified bradykinin and two novel dermaseptin-like antimicrobial peptides were fully sequenced. The novel peptide named here DMS-DA5 was fully characterized and showed potent antibacterial activity against various bacteria such as Escherichia coli, Bacillus subtilis, Salmonella enterica serovar typhimurium, and Pseudomonas aeruginosa with minimal inhibitory concentrations from 3.10 to 25.0 microM.

Analysis of mouse brain peptides using mass spectrometry-based peptidomics: implications for novel functions ranging from non-classical neuropeptides to microproteins

Peptides are known to play many important physiological roles in signaling. A large number of peptides have been detected in mouse brain extracts using mass spectrometry-based peptidomics studies, and 850 peptides have been identified. Half of these peptides are derived from secretory pathway proteins and many are known bioactive neuropeptides which activate G protein-coupled receptors; these are termed "classical neuropeptides". In addition, 427 peptides were identified that are derived from non-secretory pathway proteins; the majority are cystosolic, and the remainder are mitochondrial, nuclear, lysosomal, or membrane proteins. Many of these peptides represent the N- or C-terminus of the protein, rather than internal fragments, raising the possibility that they are formed by selective processing rather than protein degradation. In addition to consideration of the cleavage site required to generate the intracellular peptides, their potential functions are discussed. Several of the cytosolic peptides were previously found to interact with receptors and/or otherwise influence cellular activity; examples include hemorphins, hemopressins, diazepam binding inhibitor, and hippocampal cholinergic neurostimulating peptide. The possibility that these peptides are secreted from cells and function in cell-cell signaling is discussed. If these intracellular peptides can be shown to be secreted in levels sufficient to produce a biological effect, they would appropriately be called "non-classical neuropeptides" by analogy with non-classical neurotransmitters such as nitric oxide and anandamide. It is also possible that intracellular peptides function as "microproteins" and modulate protein-protein interactions; evidence for this function is discussed, along with future directions that are needed to establish this and other possible functions for peptides.

Recent advances in mass spectrometry-based peptidome analysis

The peptidome, which is the low-molecular-weight subset of the proteome, has attracted increasing attention in recent years. However, with the interference of high-abundance protein components in complex biological mixtures (e.g., serum), selective extraction of endogenous peptides is the first and most important step before analyzing the peptidome. A number of methods and technologies have now been developed for the selective enrichment, fractionation, quantitative analysis of the endogenous peptides and their application in the potential biomarker discovery. This review will cover the methods and technologies developed in recent years for the peptidome analysis on the selective extraction, multidimensional separation and quantitative analysis, as well as their application for clinical diagnosis and biomarker discovery. The future prospects of the peptidome are also discussed.