Identification Of Peptide Sequences Research Articles

A New Searching Strategy for the Identification of O-Linked Glycopeptides.

For the analysis of homogeneous post-translational modifications such as protein phosphorylation and acetylation, setting a variable modification on the specific residue(s) is applied to identify the modified peptides for database searching. However, this approach is often not applicable to identify intact mucin-type O-glycopeptides due to the high microheterogeneity of the glycosylation. Because there is virtually no carbohydrate-related tag on the peptide fragments after the O-glycopeptides are dissociated in HCD, we find it is unnecessary to set the variable mass tags on the Ser/Thr residues to identify the peptide sequences. In this study, we present a novel approach, termed as O-Search, for the interpretation of O-glycopeptide HCD spectra. Instead of setting the variable mass tags on the Ser/Thr residues, we set variable mass tags on the peptide level. The precursor mass of the MS/MS spectrum was deducted by every possible summed mass of O-glycan combinations on at most three S/T residues. All the spectra with these new precursor masses were searched against the protein sequence database without setting variable glycan modifications. It was found that this method had much decreased search space and had excellent sensitivity in the identification of O-glycopeptides. Compared with the conventional searching approach, O-Search yielded 96%, 86%, and 79% improvement in glycopeptide spectra matching, glycopeptide identification, and peptide sequence identification, respectively. It was demonstrated that O-Search enabled the consideration of more glycan structures and was fitted to analyze microheterogeneity of O-glycosylation.

Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing

BackgroundAntimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles.ResultsWe report an efficient alternative approach to selectively amplify AMP-coding transcripts from very small amounts of tissue samples, based on RNA extraction and cDNA synthesis, followed by PCR amplification and high-throughput sequencing of size-selected amplicons. This protocol exploits the highly conserved signal peptide region of the AMP precursors from Ranidae, Hylidae and Bombinatoridae for the design of family-specific, forward degenerate primers, coupled with a reverse primer targeting the mRNA poly-A tail.ConclusionsAnalysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides, mostly from Ranidae species, including several novel potential AMPs for functional characterization. This (i) confirms the effectiveness of the experimental approach and indicates points for protocol optimization to account for particular cases, and (ii) encourages the application of the same methodology to other multigenic AMP families, also from other genera, sharing common features as in anuran AMPs.

Enhanced protein identification using graphite-modified MALDI plates for offline LC-MALDI-MS/MS bottom-up proteomics

The use of offline liquid chromatography-matrix assisted laser desorption/ionization (LC-MALDI) tandem mass spectrometry (MS/MS) for bottom-up proteomics offers advantages in terms of cost, ease of use, and the time-decoupled nature of the separation step and the mass analysis. A method was developed to improve the capabilities of LC-MALDI-MS/MS in terms of protein identification in a bottom-up proteomic workflow. Enhanced protein identification is achieved by an increase in the MALDI signal intensity of the precursor peptides brought about by coating the MALDI plate with a thin film of graphite powder. Using the Escherichia coli proteome, it is demonstrated that the graphite-modified MALDI plates used in an offline LC-MALDI-MS/MS bottom-up protocol led to a 50–135% increase in the number of peptide identifications, and a concomitant 21%–105% increase in the number of proteins inferred. We identify factors that lead to improvements in peptide sequence identifications and in the number of unique proteins identified when compared to using an unmodified MALDI plate. These improvements are achieved using a low cost approach that it is easy to implement, requires no hardware/protocol modification, it is compatible with LC and adds no additional analysis time.

Identification of Functional Peptide Sequences to Lead the Design of Precision Polymers.

Peptide sciences developed dramatically as a result of routine use of solid-phase peptide synthesis and nowadays offer a rich set of well-established strategies to design and identify functional peptide sequences for advanced applications in materials sciences. Appropriate sequences for a wide range of interesting material targets, ranging from molecules to materials surfaces and internal interfaces, can be selected via combinatorial means, and sequence specificities within the resulting peptide-target interactions can be routinely investigated. Based on this understanding, macromolecular sciences can define new polymer structures that meet required functionalities or functional sequences with fully synthetic, nonpeptidic precision polymers to endeavor toward information-based design of next-generation, purpose-adapted macromolecules.

Identification of Potent ACE Inhibitory Peptides from Wild Almond Proteins.

This study has shown the potential of wild almond proteins as good sources for producing ACE-inhibitory active peptides. According to this finding, peptides with higher ACE inhibitory activities could be released during the gastrointestinal digestion and contribute to the health- promoting activities of this natural protein source.

MzStudio: A Dynamic Digital Canvas for User-Driven Interrogation of Mass Spectrometry Data.

Although not yet truly ‘comprehensive’, modern mass spectrometry-based experiments can generate quantitative data for a meaningful fraction of the human proteome. Importantly for large-scale protein expression analysis, robust data pipelines are in place for identification of un-modified peptide sequences and aggregation of these data to protein-level quantification. However, interoperable software tools that enable scientists to computationally explore and document novel hypotheses for peptide sequence, modification status, or fragmentation behavior are not well-developed. Here, we introduce mzStudio, an open-source Python module built on our multiplierz project. This desktop application provides a highly-interactive graphical user interface (GUI) through which scientists can examine and annotate spectral features, re-search existing PSMs to test different modifications or new spectral matching algorithms, share results with colleagues, integrate other domain-specific software tools, and finally create publication-quality graphics. mzStudio leverages our common application programming interface (mzAPI) for access to native data files from multiple instrument platforms, including ion trap, quadrupole time-of-flight, Orbitrap, matrix-assisted laser desorption ionization, and triple quadrupole mass spectrometers and is compatible with several popular search engines including Mascot, Proteome Discoverer, X!Tandem, and Comet. The mzStudio toolkit enables researchers to create a digital provenance of data analytics and other evidence that support specific peptide sequence assignments.

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

Identification of peptide sequences that selectively bind to pentaerythritol trinitrate hemisuccinate‐a surrogate of PETN, via phage display technology

The present research investigates the identification of amino acid sequences that selectively bind to a pentaerythritol tetranitrate (PETN) explosive surrogate. Through the use of a phage display technique and enzyme-linked immunosorbent assays (ELISA), a peptide library was tested against pentaerythritol trinitrate hemisuccinate (PETNH), a surrogate of PETN, to screen for those with amino acids having affinity toward the explosive. The results suggest that the library contains peptides selective to PETNH. Following three rounds of panning, clones were picked and tested for specificity toward PETNH. ELISA results from these samples show that each phage clone has some level of selectivity for binding to PETNH. The peptides from these clones have been sequenced and shown to contain certain common amino acid segments among them. This work represents a technological platform for identifying amino-acid sequences selective toward any bio-chem analyte of interest.

Identification of Novel Peptide Sequences with Improved Nuclear Delivery of Peptide Nucleic Acid (PNA) Sequences

Peptide Nucleic Acids, DNA mimics featuring a peptide-like backbone and nucleobases that bind nucleic acids, are capable of impacting gene expression, are resistant to degradation, and show promise as therapeutic agents. PNAs are cell-impermeant and intracellular delivery has been a challenge, however cell-penetrating peptides show some promise as delivery vehicles. To identify sequences that improve nuclear PNA delivery we constructed a library of 8192 unique peptide sequences based on the well-known cell penetrating peptides Tat and Penetratin.

N-Glycopeptide Reduction with Exoglycosidases Enables Accurate Characterization of Site-Specific N-Glycosylation

We report a new approach called NGlycoReduction that generates an oligomannosylated N-glycopeptidome by enzymatically removing sugars outside the N-glycopeptide pentasaccharide core with exoglycosidases. This approach is based on our discovery that the fragmentation of glycopeptides is glycan-structure dependent and glycans with core mannose structures overwhelmingly lead to the generation of Y1 ions when subjected to MS/MS in mass spectrometry. Oligomannosylated glycopeptidome produced by NGlycoReduction can be mixed with the intact N-glycopeptidome and analyzed by HPLC-ESI-MS together to enable the identification of peptide sequence, glycosylation site and the structure of intact glycopeptides. The glycan structure of intact glycopeptides can be identified from MS/MS spectra of their own and their peptide sequences were identified by the MS3 spectra of the oligomannosylated glycopeptides with the same Y1 ion. Both mass tolerance and difference in retention time were further used to increase the confidence in the Y1 ion alignment. This approach has the advantage of low cost and ease of processing and can be expanded to other samples, especially for characterizing site-specific N-glycosylation involving complex N-glycans. In this study, simultaneous analysis of the combined oligomannosylated N-glycopeptidome and the native glycopeptidome leads to the identification of 609 N-glycopeptides from the secretome and lysates of Huh7 cells.

A rapid and sensitive detection of D-Aspartic acid in Crystallin by chiral derivatized liquid chromatography mass spectrometry

A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI–MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD<0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC–MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp76 and Asp84 in the αA-Crystallin and Asp96 in αB-Crystallin were racemized to D-Asp. These results show that this proposed chiral peptide LC–MS/MS method using chiral derivatization provides a rapid and sensitive analysis for post translational Asp racemization sites in aging proteins.

Parallel data acquisition of in-source fragmented glycopeptides to sequence the glycosylation sites of proteins.

Glycosylation plays important roles in maintaining protein stability and controlling biological processes. In recent years, the correlation between aberrant glycoproteins and many diseases has been reported. Hence, qualitative and quantitative analyses of glycoproteins are necessary to understand physiological processes. LC-MS/MS analysis of glycopeptides is faced with the low glycopeptide signal intensities and low peptide sequence identification. In our study, in-source fragmentation (ISF) was used in conjunction with LC-MS/MS to facilitate the parallel acquisition of peptide backbone sequence and glycan composition information. In ISF method, the identification of glycosylation sites depended on the detection of Y1 ion (ion of peptide backbone with an N-acetylglucosamine attached). To attain dominant Y1 ions, a range of source fragmentation voltages was studied using fetuin. A 45 V ISF voltage was found to be the most efficient voltage for the analysis of glycoproteins. ISF was employed to study the glycosylation sites of three model glycoproteins, including fetuin, α1-acid glycoprotein and porcine thyroglobulin. The approach was then used to analyze blood serum samples. Y1 ions of glycopeptides in tryptic digests of samples were detected. Y1 ions of glycopeptides with different sialic acid groups are observed at different retention times, representing the various numbers of sialic acid moieties associated with the same peptide backbone sequence. With ISF facilitating the peptide backbone sequencing of glycopeptides, identified peptide sequence coverage was increased. For example, identified fetuin sequence percentage was improved from 39 to 80% in MASCOT database searching compared to conventional CID method. The formation of Y1 ions and oxonium ions in ISF facilitates glycopeptide sequencing and glycan composition identification.

Application of de Novo Sequencing to Large-Scale Complex Proteomics Data Sets.

Dependent on concise, predefined protein sequence databases, traditional search algorithms perform poorly when analyzing mass spectra derived from wholly uncharacterized protein products. Conversely, de novo peptide sequencing algorithms can interpret mass spectra without relying on reference databases. However, such algorithms have been difficult to apply to complex protein mixtures, in part due to a lack of methods for automatically validating de novo sequencing results. Here, we present novel metrics for benchmarking de novo sequencing algorithm performance on large-scale proteomics data sets and present a method for accurately calibrating false discovery rates on de novo results. We also present a novel algorithm (LADS) that leverages experimentally disambiguated fragmentation spectra to boost sequencing accuracy and sensitivity. LADS improves sequencing accuracy on longer peptides relative to that of other algorithms and improves discriminability of correct and incorrect sequences. Using these advancements, we demonstrate accurate de novo identification of peptide sequences not identifiable using database search-based approaches.

Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate

Short peptides in food protein hydrolysates are of significant interest as they may be highly bioactive whilst also being bioavailable. A dipeptidyl peptidase IV (DPP-IV) inhibitory whey protein hydrolysate (WPH) was fractionated using nanofiltration (NF) with a 200Da MWCO membrane. The DPP-IV half maximal inhibitory concentration of the NF permeate (IC50=0.66±0.08mgprotein equivalentmL−1) was significantly more potent (P>0.05) than that of the starting WPH (IC50=0.94±0.24mgprotein equivalentmL−1) and associated retentate (IC50=0.82±0.13mgprotein equivalentmL−1). This confirmed the contribution of short peptides within the NF permeate to the overall DPP-IV inhibitory activity. An hydrophilic interaction liquid chromatography (HILIC-) and reverse-phase (RP-) liquid chromatography tandem mass spectrometry (LC–MS/MS) strategy, based on two retention time models, allowed detection of eight free amino acids and eight di- to tetrapeptides in the NF permeate. The potential sequences of the peptides within the NF permeate were then ranked on the basis of their highest probability of occurrence. A confirmatory study with synthetic peptides showed that valine–alanine (VA), valine–leucine (VL), tryptophan–leucine (WL) and tryptophan–isoleucine (WI) displayed DPP-IV IC50 values <170μM. The NF and LC–MS strategies employed herein represent a new approach for the targeted identification of short peptides within bioactive food protein hydrolysates.

Identification of short peptide sequences in complex milk protein hydrolysates

Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates.

Identification of Bioactive Peptide Sequences from Amaranth (Amaranthus hypochondriacus) Seed Proteins and Their Potential Role in the Prevention of Chronic Diseases.

Amaranth (Amaranthus hypochondriacus) is a pseudocereal with higher protein concentration than most cereal grains. Enzymatic hydrolysis and food processing could produce biopeptides from amaranth proteins; however, there is limited information about the bioactivity of peptides from amaranth proteins. The objective of this comprehensive review was to determine bioactive peptide sequences in amaranth proteins that may prevent cardiovascular disease, cancer, and diabetes. Amaranth proteins, reported in UniProt database, were evaluated for potential bioactive peptide using BIOPEP database. The 15 main proteins present in amaranth seed are 11S globulin, 7S globulin, α-amylase inhibitor, trypsin inhibitor, antimicrobial proteins, nonspecific lipid-transfer-protein-1, superoxide dismutase, ring-zinc finger protein, prosystemin, amaranth albumin 1, glucose-1-phosphate adenyltransferase, glucosyltransferase, polyamine oxidase, granule-bound starch synthase 1,and acetolactate synthase. All proteins showed high occurrence frequencies of angiotensin-converting enzyme-inhibitor peptides (A = 0.161 to 0.362), as well as of dipeptidyl peptidase IV inhibitor (A = 0.003 to 0.087). Other proteins showed antioxidative (A = 0.012 to 0.063) and glucose uptake-stimulating activity (A = 0.023 to 0.042), and also antithrombotic (A = 0.002 to 0.031) and anticancer sequences (A = 0.001 to 0.042). The results of this study support the concept that amaranth grain could be part of a "healthy" diet and thereby prevent chronic human diseases.

MAGIC: an automated N-linked glycoprotein identification tool using a Y1-ion pattern matching algorithm and in silico MS² approach.

Glycosylation is a highly complex modification influencing the functions and activities of proteins. Interpretation of intact glycopeptide spectra is crucial but challenging. In this paper, we present a mass spectrometry-based automated glycopeptide identification platform (MAGIC) to identify peptide sequences and glycan compositions directly from intact N-linked glycopeptide collision-induced-dissociation spectra. The identification of the Y1 (peptideY0 + GlcNAc) ion is critical for the correct analysis of unknown glycoproteins, especially without prior knowledge of the proteins and glycans present in the sample. To ensure accurate Y1-ion assignment, we propose a novel algorithm called Trident that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0-NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides. To facilitate the subsequent peptide sequence identification by common database search engines, MAGIC generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions. Finally, MAGIC computes the glycan compositions and ranks them. For the model glycoprotein horseradish peroxidase (HRP) and a 5-glycoprotein mixture, a 2- to 31-fold increase in the relative intensities of the peptide fragments was achieved, which led to the identification of 7 tryptic glycopeptides from HRP and 16 glycopeptides from the mixture via Mascot. In the HeLa cell proteome data set, MAGIC processed over a thousand MS(2) spectra in 3 min on a PC and reported 36 glycopeptides from 26 glycoproteins. Finally, a remarkable false discovery rate of 0 was achieved on the N-glycosylation-free Escherichia coli data set. MAGIC is available at http://ms.iis.sinica.edu.tw/COmics/Software_MAGIC.html .

The Potential of Proteomics in Understanding Neurodegeneration.

Neurodegenerative diseases are a major health concern worldwide. Diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as many other diseases affecting the neuromuscular system, are a leading cause of disability in the aging population. Presymptomatic diagnosis of neurodegenerative disorders is challenging due to the lack of robust biomarkers. Likewise, the design of effective intervention strategies is limited because most neurodegenerative disorders are heterogeneous in nature. Reliable noninvasive biomarkers are therefore urgently needed to allow presymptomatic and accurate diagnosis, to track disease progression, to evaluate the effectiveness of new treatment regimens, and to ultimately design new therapeutic intervention strategies. Recent biological and technological advances within the field of proteomic promises to provide insight into global proteome changes in neurodegeneration, thus allowing increased understanding of molecular pathways leading to neuronal cell death and the identification of biomarkers. The combination of gel-based techniques and mass spectrometry permits large-scale identification of peptide sequences in biological samples as well as the characterization of posttranslational protein modifications. The application of comparative high-throughput proteomic analyses in animal models and human tissues will aid in the identification of both diagnostic and prognostic biomarkers and will provide a platform for a future personalized medicine approach in neurodegeneration.

Research and Development of a Peptide Complex Technology

Results of biotechnological research on controlled hydrolysis of casein and production of peptide complexes are summarized in the present article. Selection of processing parameters and optimization of the conditions of enzymatic hydrolysis of milk proteins allowed for the development of a resource-efficient technology for the production of peptide complexes. Enzymatic hydrolysis of peptide bonds R1-CONH-R2 + H2O  R1COOH + NH2R2 was considered using the example of casein and a range of proteolytic enzymes (trypsin and chymotrypsin) belonging to the hydrolase class. Enzyme solution (0.1% m/v) was added to each protein solution so that the final enzymesubstrate ratio was 1:25, 1:50, or 1:100. The enzyme-substrate ratio of 1:50 was shown to be optimal, and the recommended temperature and pH values were 50±1°С and 7.50±0.01, respectively. The degree of hydrolysis is one of the parameters characterizing the overall changes in the amino acid composition of proteins. Therefore, a time period of 12.00±0.05 h was chosen as the optimal duration of the hydrolysis process. Further research was focused on the analysis of peptide profiles using MALDI-TOF MS based on identification of peptide sequences. The studies have shown that casein hydrolysates are rich in biologically active peptide complexes. The detection of such complexes in hydrolysates of casein was the main result of the study. For example, the peptide β-casokinin (amino acid sequence Ala-Val-Pro-Tyr-Pro-Gln-Arg) is an inhibitor of angiotensin-converting enzyme.

MS Amanda, a universal identification algorithm optimized for high accuracy tandem mass spectra.

Today’s highly accurate spectra provided by modern tandem mass spectrometers offer considerable advantages for the analysis of proteomic samples of increased complexity. Among other factors, the quantity of reliably identified peptides is considerably influenced by the peptide identification algorithm. While most widely used search engines were developed when high-resolution mass spectrometry data were not readily available for fragment ion masses, we have designed a scoring algorithm particularly suitable for high mass accuracy. Our algorithm, MS Amanda, is generally applicable to HCD, ETD, and CID fragmentation type data. The algorithm confidently explains more spectra at the same false discovery rate than Mascot or SEQUEST on examined high mass accuracy data sets, with excellent overlap and identical peptide sequence identification for most spectra also explained by Mascot or SEQUEST. MS Amanda, available at http://ms.imp.ac.at/?goto=msamanda, is provided free of charge both as standalone version for integration into custom workflows and as a plugin for the Proteome Discoverer platform.