Proteases are widely used in the food industry to hydrolyze proteins and prepare bioactive peptides. Peptide mapping identification supports the application of proteases in the food industry. The site-specified peptide identification method, which was developed for site-specific proteases like trypsin, is relatively mature and reliable but cannot be applied using most industrial proteases with weak site specificity. To address this issue, the performance and reliability of the site-unspecified peptide identification method should be investigated and evaluated. In this study, tryptic hydrolysates of a single protein and a protein mixture were used to evaluate the site-unspecified identification method. The species origin of the hydrolyzed proteins was not specified in a database search, meaning that millions of protein sequences were included for calculating and matching. At least 98% of the tryptic peptides were successfully identified via the site-unspecified method, demonstrating that the site-unspecified method shows promising reliability. Moreover, the site-unspecified method identified more peptides than the site-specified method, including those from the low-frequency site-unspecific hydrolysis of trypsin, suggesting that the method has strong capabilities for peptide mapping. The results indicate the applicability of the site-unspecified peptide identification method in the study of site-unspecific industrial proteases.

Neuropeptides and peptide hormones are ancient, widespread signaling molecules that underpin almost all brain functions. They constitute a broad ligand-receptor network, mainly by binding to G protein-coupled receptors (GPCRs). However, the organization of the peptidergic network and roles of many peptides remain elusive, as our insight into peptide-receptor interactions is limited and many peptide GPCRs are still orphan receptors. Here we report a genome-wide peptide-GPCR interaction map in Caenorhabditis elegans. By reverse pharmacology screening of over 55,384 possible interactions, we identify 461 cognate peptide-GPCR couples that uncover a broad signaling network with specific and complex combinatorial interactions encoded across and within single peptidergic genes. These interactions provide insights into peptide functions and evolution. Combining our dataset with phylogenetic analysis supports peptide-receptor co-evolution and conservation of at least 14 bilaterian peptidergic systems in C. elegans. This resource lays a foundation for system-wide analysis of the peptidergic network.

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

Identification of peptides mediating protein-protein interaction (PPI) is crucial for understanding the function of interlinked proteins in cellular processes and amino acid-associated diseases. Traditional PPI assays are laborious, involving the generation of many truncated proteins. SPOT peptide assay allows high-throughput detection of domains essential for PPI by synthesizing several hundred peptides on a cellulose membrane. Here, we present a rapid SPOT peptide protocol for identifying the binding motifs, which mediate interaction between the chromatin remodeling factors BAF155/BAF170 and the epigenetic factor Kdm6b. For complete details on the use and execution of this protocol, please refer to Narayanan et al. (2015).

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure. Graphical abstract Peptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data.

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.

In India, Sorghum plant allergenicity was reported to be approximately 54.9%. Sorghum bicolor Polcalcin (Sorb PC) was identified as the panallergen but the specificity of this allergen is yet to be characterized. The present study was aimed to characterize the antigenic determinants of Sorb PC that are responsible for eliciting the IgE response. In silico modeling, simulation studies and docking of Sorb PC peptides (PC1-11) against IgG and IgE followed by in vivo evaluation was adopted. Peptide docking studies revealed PC 6 with highest G-score -12.85 against IgE followed by PC-11, 5, 1 and 7 (-10.91) peptides. The mice sensitized with PC7 peptide showed interleukin (IL) 4 (IL-4), IL-5, IL-12, TNF-α and GMCSF levels increased when compared with other peptides and controls, signifying a strong T helper type 2 (Th2)-based response. In tandem, the T helper type 1 (Th1) pathway was inhibited by low levels of cytokine IL-2, interferon γ (IFN-γ) and increased IL-10 levels justifying the role of PC7 in allergic IgE response. Considering the above data of overlapping peptides of PC6 and PC7, N-terminal part of the PC7 peptide (DEVQRMM) is found to play a crucial role in Sorghum Polcalcin allergenic response.

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections.

The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides.

One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery.

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

Nearly 20-30% of the world's population suffers from allergic rhinitis, among them 15% are progressing to asthma conditions. Sorghum bicolor profilin (Sorb PF), one of the panallergens, was identified, but the allergen specificity is not yet characterized.

Immunological reactivity of partially purified hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from brain of several vertebrate species has been compared by using enzyme-linked immunosorbent assay and seven monoclonal antibodies raised against the rat brain enzyme. The epitopes recognized by three of these antibodies have been rather widely conserved among the species examined (rat, mouse, guinea pig, rabbit, cat, dog, sheep, cow, pig, chicken), while this was not the case for the epitopes recognized by the other antibodies, which differed markedly in their distribution among these species. The domain structure of these enzymes has been examined by peptide mapping (after limited tryptic digestion) in conjunction with immunoblotting techniques employing monoclonal antibodies. The results indicate that the overall domain structure of these enzymes is similar to that previously described for rat brain hexokinase A, but that there are significant differences in the size of these domains in enzymes from different species.

Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape both delineate pathways of resistance to these fusion peptide-directed antibodies and provide a strategy to improve the breadth or potency of future vaccine-induced antibodies against Env's fusion peptide.

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus.

The voltage-gated sodium (Nav) channel complex is comprised of pore-forming α subunits (Nav1.1-1.9) and accessory regulatory proteins such as the intracellular fibroblast growth factor 14 (FGF14). The cytosolic Nav1.6 C-terminal tail binds directly to FGF14 and this interaction modifies Nav1.6-mediated currents with effects on intrinsic excitability in the brain. Previous studies have identified the FGF14V160 residue within the FGF14 core domain as a hotspot for the FGF14:Nav1.6 complex formation. Here, we used three short amino acid peptides around FGF14V160 to probe for the FGF14 interaction with the Nav1.6 C-terminal tail and to evaluate the activity of the peptide on Nav1.6-mediated currents. In silico docking predicts FLPK to bind to FGF14V160 with the expectation of interfering with the FGF14:Nav1.6 complex formation, a phenotype that was confirmed by the split-luciferase assay (LCA) and surface plasmon resonance (SPR), respectively. Whole-cell patch-clamp electrophysiology studies demonstrate that FLPK is able to prevent previously reported FGF14-dependent phenotypes of Nav1.6 currents, but that its activity requires the FGF14 N-terminal tail, a domain that has been shown to contribute to Nav1.6 inactivation independently from the FGF14 core domain. In medium spiny neurons in the nucleus accumbens, where both FGF14 and Nav1.6 are abundantly expressed, FLPK significantly increased firing frequency by a mechanism consistent with the ability of the tetrapeptide to interfere with Nav1.6 inactivation and potentiate persistent Na+ currents. Taken together, these results indicate that FLPK might serve as a probe for characterizing molecular determinants of neuronal excitability and a peptide scaffold to develop allosteric modulators of Nav channels.

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.