Peptidomics: a logical sequel to proteomics

Rapid progress of separation techniques as well as methods of structural analysis provided conditions in the past decade for total screening of complex biologic mixtures for any given class of biomolecules. The present review updates the reader with the modern state of peptidomics, a chapter of chemical biology that deals with structure and biologic properties of sets of peptides present in biologic tissues, cells or fluids. Scope and limitations of currently employed experimental techniques are considered and the main results are outlined. Considerable attention will be afforded to the biologic role of peptides formed in vivo by proteolysis of nonspecialized precursor proteins with other well-defined functions. In conclusion, the connection is discussed between peptidomics and the much more mature and still closely related field of proteomics.     

Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery

The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology.Proteomics technologies have evolved to produce larg...     

Peptidomic analysis of blood plasma after in vivo treatment with protease inhibitors--a proof of concept study

Native peptides can be regarded as surrogate markers for protease activity in biological samples. Analysis of peptides by peptidomics allows to monitor protease activity in vivo and to describe the influence of protease inhibition. To elucidate the potential of peptides as markers for in vivo protease inhibition we analyzed plasma samples from animals treated with either the indirect FXa inhibitor FONDAPARINUX™ or the dipeptidylpeptidase IV inhibitor AB192. Signals correlating with the treatment were subsequently identified and assessed with respect to protease-dependent consensus cleavage motifs and occurrence of downstream targets. It could be shown that regulated peptides were either substrates, products or downstream targets of the inhibited protease. The results from the present study demonstrate that the in vivo analysis of peptides by peptidomics has the potential to broaden the knowledge of inhibitor related effects in vivo and that this method may pave the way to develop predictive biomarkers.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae)

In this study, peptidomics and genomics analyses were used to study antimicrobial peptides from the skin of Hylarana spinulosa. Twenty-nine different antimicrobial peptide precursors were characterized from the skin of H. spinulosa, which produce 23 mature antimicrobial peptides belonging to 12 different families. To confirm the actual presence and characteristics of these antimicrobial peptides in the skin tissue extractions from H. spinulosa, we used two distinct methods, one was peptide purification method that combined gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the other was peptidomics approach based on liquid chromatography in conjunction with tandem mass spectrometry (LC–MS/MS). In the peptidomics approach, incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase peptidome coverage and reproducibility of peptide ion selection. Multiple species of microorganisms were chosen to test and analyze the antimicrobial activities and spectrum of these antimicrobial peptides.     

Digging Deep into Peptidomics Applied to Body Fluids

Peptidomics techniques have allowed the identification of thousands of peptides that are derived from proteins in body fluids, despite the considerable challenges behind sample handling, MS‐based identification, data analysis, and integration with bioinformatics tools. Body fluids’ naturally occurring peptides are known to perform a variety of local and systemic functions; however, its knowledge is limited. Even so, the biological meaning that can be retrieved from peptidomics applied to the identification of disease markers and to the development of therapies using peptides has driven the progresses made in this field. In this review, a comparative analysis of body fluids’ peptidome data retrieved from databases and from scientific papers is performed to identify the biological processes modulated by naturally occurring peptides. This integrative analysis highlights several interesting facts, such as the small overlap between blood‐derived serum and plasma, which illustrates the impact of sample handling on these fluids peptidome. Urine is the body fluid with more naturally occurring peptides identified so far, most of which are derived from collagens. In saliva, the majority of peptides are originated from extracellular matrix proteins. Cerebrospinal fluid presents a high number of peptides derived from distinct proteins, mostly involved in the regulation of nervous system homeostasis. The lowest number of endogenous peptides was found in tears, most of which present antimicrobial activity. Collectively, data analysis highlights a peptidome signature for each body fluid, which comprehension will certainly help to improve disease management.     

Profiling thimet oligopeptidase-mediated proteolysis in Arabidopsis thaliana

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.     

Application of a Combined Peptidomics and In Silico Approach for the Identification of Novel Dipeptidyl Peptidase-IV-Inhibitory Peptides in In Vitro Digested Pinto Bean Protein Extract

The conventional approach in bioactive peptides discovery, which includes extensive bioassay-guided fractionation and purification processes, is tedious, time-consuming and not always successful. The recently developed bioinformatics-driven in silico approach is rapid and cost-effective; however, it lacks an actual physiological significance. In this study a new integrated peptidomics and in silico method, which combines the advantages of the conventional and in silico approaches by using the pool of peptides identified in a food hydrolysate as the starting point for subsequent application of selected bioinformatics tools, has been developed. Pinto bean protein extract was in vitro digested and peptides were identified by peptidomics. The pool of obtained peptides was screened by in silico analysis and structure–activity relationship modelling. Three peptides (SIPR, SAPI and FVPH) were selected as potential inhibitors of the dipeptidyl-peptidase-IV (DPP-IV) enzyme by this integrated approach. In vitro bioactivity assay showed that all three peptides were able to inhibit DPP-IV with the tetra-peptide SAPI showing the highest activity (IC50 = 57.7 μmol/L). Indeed, a new possible characteristic of peptides (i.e., the presence of an S residue at the N-terminus) able to inhibit DPP-IV was proposed.     

Identifying a urinary peptidomics profile for hypertension in young adults: The African-PREDICT study

Hypertension is one of the most important and complex risk factors for cardiovascular diseases (CVDs). By using urinary peptidomics analyses, we aimed to identify peptides associated with hypertension, building a framework for future research towards improved prediction and prevention of premature development of CVD. We included 78 hypertensive and 79 normotensive participants from the African-PREDICT study (aged 20–30 years), matched for sex (51% male) and ethnicity (49% black and 51% white). Urinary peptidomics data were acquired using capillary-electrophoresis-time-of-flight-mass-spectrometry. Hypertension-associated peptides were identified and combined into a support vector machine-based multidimensional classifier. When comparing the peptide data between the normotensive and hypertensive groups, 129 peptides were nominally differentially abundant (Wilcoxon p < 0.05). Nonetheless, only three peptides, all derived from collagen alpha-1(III), remained significantly different after rigorous adjustments for multiple comparisons. The 37 most significant peptides (all p ≤ 0.001) served as basis for the development of a classifier, with 20 peptides being combined into a unifying score, resulting in an AUC of 0.85 in the ROC analysis (p < 0.001), with 83% sensitivity at 80% specificity. Our study suggests potential value of urinary peptides in the classification of hypertension, which could enable earlier diagnosis and better understanding of the pathophysiology of hypertension and premature cardiovascular disease development.     

Combinatorial peptidomics: a generic approach for protein expression profiling

Traditional approaches to protein profiling were built around the concept of investigating one protein at a time and have long since reached their limits of throughput. Here we present a completely new approach for comprehensive compositional analysis of complex protein mixtures, capable of overcoming the deficiencies of current proteomics techniques. The Combinatorial methodology utilises the peptidomics approach, in which protein samples are proteolytically digested using one or a combination of proteases prior to any assay being carried out. The second fundamental principle is the combinatorial depletion of the crude protein digest (i.e. of the peptide pool) by chemical crosslinking through amino acid side chains. Our approach relies on the chemical reactivities of the amino acids and therefore the amino acid content of the peptides (i.e. their information content) rather than their physical properties. Combinatorial peptidomics does not use affinity reagents and relies on neither chromatography nor electrophoretic separation techniques. It is the first generic methodology applicable to protein expression profiling, that is independent of the physical properties of proteins and does not require any prior knowledge of the proteins. Alternatively, a specific combinatorial strategy may be designed to analyse a particular known protein on the basis of that protein sequence alone or, in the absence of reliable protein sequence, even the predicted amino acid translation of an EST sequence. Combinatorial peptidomics is especially suitable for use with high throughput micro- and nano-fluidic platforms capable of running multiple depletion reactions in a single disposable chip.     

Peptidomics in Drosophila melanogaster.

In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analysed, the peptidomic approach aims at the simultaneous visualisation and identification of the whole peptidome of a cell or tissue, ie all expressed peptides with their post-translational modifications. With nanoscale liquid chromatography (nanoLC), combined with mass spectrometry and subsequent database searching, the peptidome of the Drosophila larval brain has been identified at the amino acid sequence level. In a single experiment involving only 50 Drosophila larval brains, one can obtain a display of the expressed peptides. In this paper, current peptidomics technology will be explained, using Drosophila as an example. Compared with the 400,000 Drosophila whole bodies that were required as a starting material for traditional biochemical peptide purification rounds, the authors are convinced that peptidomics technology, which in the future will certainly be applied to the analysis of different physiological states, has the inherent potential to bring about a true revolution in the study of the molecular physiology of Drosophila.     

Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe^fat/fat mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe^fat/fat mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe^fat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe^fat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe^fat/fat mice; these peptide processing intermediates with C‐terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.     

The zebra finch neuropeptidome: prediction,detection and expression

Background  

Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions.     

Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reaction

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.     

Diapausing Colorado potato beetles are devoid of short neuropeptide F I and II

An extract of head ganglia and retrocerebral complexes of nondiapausing and diapausing Leptinotarsa decemlineata was prepared to characterize regulatory neuropeptides involved in adult diapause by using a differential peptidomics approach. To reduce sample complexity, both extracts were roughly separated by means of an identical chromatographic step. MALDI-TOF MS led to the identification of proctolin, an adipokinetic hormone, and short neuropeptide F I and II in the extract of nondiapausing beetles. In combination with nano-ESI-Q-TOF MS(2) evidence was found for the presence of three pyrokinins, the first to be identified in a coleopteran species. Pyrokinins, involved in the induction of embryonic diapause in Bombyx mori, were present in both physiological conditions suggesting that they are of minor importance in the regulation of adult diapause in the Colorado potato beetle. A striking difference, detected by the differential peptidomics approach, between both neuropeptide profiles was the absence of ions corresponding to the short neuropeptide F (sNPF) related peptides, also known as Led-NPF-I and -II, in the extract of diapausing animals. Therefore, we postulate that "short NPFs" are involved in the regulation of adult diapause, displayed by the Colorado potato beetle.     

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.     

Peptidomic analysis of the skin secretions of the frog <Emphasis Type="Italic">Pachymedusa dacnicolor</Emphasis>

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 μM.     

Plant peptides and peptidomics

Extracellular plant peptides perform a large variety of functions, including signalling and defence. Intracellular peptides often have physiological functions or may merely be the products of general proteolysis. Plant peptides have been identified and, in part, functionally characterized through biochemical and genetic studies, which are lengthy and in some cases impractical. Peptidomics is a branch of proteomics that has been developed over the last 5 years, and has been used mainly to study neuropeptides in animals and the degradome of proteases. Peptidomics is a fast, efficient methodology that can detect minute and transient amounts of peptides and identify their post-translational modifications. This review describes known plant peptides and introduces the use of peptidomics for the detection of novel plant peptides.     

Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.