Majority Of Peptides Research Articles

Influence of Software Settings on the Identification Rate, Quantification Results, and Reproducibility in Profiling Post-Translational Modifications by Microflow Liquid Chromatography-Ion Mobility-Quadrupole Time-Of-Flight Analysis Using PEAKS Software.

The influence of data evaluation parameters on qualitative and quantitative results of untargeted shotgun profiling of enzymatic and nonenzymatic post-translational modifications (PTMs) was investigated in a model of bovine whey protein α-lactalbumin heated with lactose. Based on the same raw data, individual adjustments to the protein database and enzyme settings of PEAKS studio software increased the identification rate from 27 unmodified peptides to 48 and from 322 peptides in total to 535. The qualitative and quantitative reproducibility was also assessed based on 18 measurements of one sample across three batches. A total of 570 peptides were detected. While 89 peptides were identified in all measurements, the majority of peptides (161) were detected only once and mostly based on nonindicative spectra. The reproducibility of label-free quantification (LFQ) in six measurements of the same sample was similar after processing the data by either the PTM algorithm or the LFQ algorithm. In both cases, about one-third of the peptides showed a coefficient of variation of above 20%. However, the LFQ algorithm increased the number of quantified peptides from 75 to 179. Data are available at the PRIDE Archive with the data set identifier PXD050363.

Diversity analysis of sea anemone peptide toxins in different tissues of Heteractis crispa based on transcriptomics

Peptide toxins found in sea anemones venom have diverse properties that make them important research subjects in the fields of pharmacology, neuroscience and biotechnology. This study used high-throughput sequencing technology to systematically analyze the venom components of the tentacles, column, and mesenterial filaments of sea anemone Heteractis crispa, revealing the diversity and complexity of sea anemone toxins in different tissues. A total of 1049 transcripts were identified and categorized into 60 families, of which 91.0% were proteins and 9.0% were peptides. Of those 1049 transcripts, 416, 291, and 307 putative proteins and peptide precursors were identified from tentacles, column, and mesenterial filaments respectively, while 428 were identified when the datasets were combined. Of these putative toxin sequences, 42 were detected in all three tissues, including 33 proteins and 9 peptides, with the majority of peptides being ShKT domain, β-defensin, and Kunitz-type. In addition, this study applied bioinformatics approaches to predict the family classification, 3D structures, and functional annotation of these representative peptides, as well as the evolutionary relationships between peptides, laying the foundation for the next step of peptide pharmacological activity research.

Revealing Natural Intracellular Peptides in Gills of Seahorse Hippocampus reidi.

The seahorse is a marine teleost fish member of the Syngnathidae family that displays a complex variety of morphological and reproductive behavior innovations and has been recognized for its medicinal importance. In the Brazilian ichthyofauna, the seahorse Hippocampus reidi is among the three fish species most used by the population in traditional medicine. In this study, a protocol was performed based on fast heat inactivation of proteases plus liquid chromatography coupled to mass spectrometry to identify native peptides in gills of seahorse H. reidi. The MS/MS spectra obtained from gills allowed the identification of 1080 peptides, of which 1013 peptides were present in all samples and 67 peptide sequences were identified in an additional LC-MS/MS run from an alkylated and reduced pool of samples. The majority of peptides were fragments of the internal region of the amino acid sequence of the precursor proteins (67%), and N- and C-terminal represented 18% and 15%, respectively. Many peptide sequences presented ribosomal proteins, histones and hemoglobin as precursor proteins. In addition, peptide fragments from moronecidin-like protein, described with antimicrobial activity, were found in all gill samples of H. reidi. The identified sequences may reveal new bioactive peptides.

Peptidomics-based identification of an antimicrobial peptide derived from goat milk fermented by Lactobacillus rhamnosus (C25).

Antimicrobial peptides (AMPs) are emerging as promising novel drug applicants. In the present study, goat milk was fermented using Lactobacillus rhamnosus C25 to generate bioactive peptides (BAPs). The peptide fractions generated were separated using ultrafiltration membranes with molecular weight cut-offs of 3, 5, and 10kDa, and their antimicrobial activity toward Gram-positive and Gram-negative bacteria was investigated. Isolated AMPs were characterized using RP-HPLC and identified by LC-MS/MS. A total of 569 sequences of peptides were identified by mass spectrometry. Out of the 569, 36 were predicted as AMPs, 21 were predicted as cationic, and out of 21, 6 AMPs were helical peptides. In silico analysis indicated that the majority of peptides were antimicrobial and cationic in nature, an important factor for peptide interaction with the negative charge membrane of bacteria. The results showed that the peptides of <5kDa exhibited maximum antibacterial activity against E. faecalis, E. coli, and S. typhi. Further, molecular docking was used to evaluate the potent MurD ligase inhibitors. On the basis of ligand binding energy, six predicted AMPs were selected and then analyzed by AutoDock tools. Among the six AMPs, peptides IGHFKLIFSLLRV (-7.5kcal/mol) and KSFCPAPVAPPPPT (-7.6kcal/mol), were predicted as a high-potent antimicrobial. Based on these findings, in silico investigations reveal that proteins of goat milk are a potential source of AMPs. This is for the first time that the antimicrobial peptides produced by Lactobacillus rhamnosus (C25) fermentation of goat milk have been identified via LC-MS/MS and predicted as AMPs, cationic charges, helical structure in nature, and potent MurD ligase inhibitors. These peptides can be synthesized and improved for use as antimicrobial agents. PRACTICAL APPLICATIONS: Goat milk is considered a high-quality source of milk protein. According to this study, goat milk protein is a potential source of AMPs, Fermentation can yield goat milk-derived peptides with a broad antibacterial activity spectrum at a low cost. The approach described here could be beneficial in that the significant AMPs can be synthesized and used in the pharmaceutical and food industries.

Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed

In this work, we sequentially extracted water (CSPw)- and alkali (CSPa)-soluble protein fractions from glandless cottonseed. SDS-Gel electrophoresis separated CSPw and CSPa to 8 and 14 dominant polypeptide bands (110–10 kDa), respectively. Liquid chromatography-electrospray ionization-tandem mass spectrometry identified peptide fragments from 336 proteins. While the majority of peptides were identified as belonging to vicilin and legumin storage proteins, peptides from other functional and uncharacterized proteins were also detected. Based on the types (unique peptide count) and relative abundance (normalized total ion current) of the polypeptides detected by mass spectrometry, we found lower levels (abundance) and types of legumin isoforms, but higher levels and more fragments of vicilin-like antimicrobial peptides in glandless samples, compared to glanded samples. Differences in peptide fragment patterns of 2S albumin and oleosin were also observed between glandless and glanded protein samples. These differences might be due to the higher extraction recovery of proteins from glandless cottonseed as proteins from glanded cottonseed tend to be associated with gossypol, reducing extraction efficiency. This work enriches the fundamental knowledge of glandless cottonseed protein composition. For practical considerations, this peptide information will be helpful to allow better understanding of the functional and physicochemical properties of glandless cottonseed protein, and improving the potential for food or feed applications.

Gastric Digestion of Milk Proteins in Adult and Elderly: Effect of High-Pressure Processing.

Reduced physiological capability of the human gastrointestinal tract with increasing age has recently attracted considerable attention to the potential of novel technologies to modify food digestion. Thus, the aim of this study was to investigate gastric digestion of milk proteins after application of high-pressure processing (HPP) at 400 MPa 15 min, 600 MPa 5 min and 600 MPa 15 min using two static in vitro models of adults (INFOGEST) and the elderly in comparison to a fresh untreated raw milk. Peptides distribution classified based on the number of amino acids (AA) (<10, 11–15, 16–20, 21–30, >30 AA) were investigated after 0, 5, 10 and 30 min of digestion using LC–MS and multivariate data analysis. Our results show significantly less efficient protein digestion of all investigated milks in the elderly model indicated by higher percentages of longer peptides during digestion, except for the HPP milk 400 MPa 15 min, which indicated an improved and comparable digestion in the elderly as in the adult model. Furthermore, increasing the pressurization time at 600 MPa did not have a significant effect on the peptides profile during the digestion. More efficient digestion of whey proteins in HPP milks, with the majority of peptides in the 16–20 AA range, compared to fresh milk was also noticed. According to the findings of this study, HPP at 400 MPa 15 min showed the most efficient digestion of major milk proteins and thus may be considered a suitable process to improve bioaccessibility of milk proteins, especially in products intended for the elderly.

Trending Anti-Aging Peptides

The development of synthetic peptides for skin care dates to the 1980s. The cosmetic industry periodically launches new peptides, as they are promising and appealing active ingredients in the growing and innovative cosmetics market. In this study, trends in the use of peptides in anti-aging products were analyzed by comparing the composition of the products marketed in 2011 with products launched or reformulated in 2018. The scientific and marketing evidence for their application as active ingredients in anti-aging cosmetics was also compiled from products’ labels, suppliers’ technical data forms and online scientific databases. The use of peptides in anti-aging cosmetics increased by 7.2%, while the variety and the number of peptide combinations in products have increased by 88.5%. The most used peptides in antiaging cosmetic formulations are, in descending order, Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide and Acetyl Hexapeptide-8. In 2011, the majority of peptides were obtained from synthesis, while in 2018, biotechnology processing was the dominant source. This study provides an overview of the market trends regarding the use of peptides in anti-aging products, providing meaningful data for scientists involved in the development of new peptides to identify opportunities for innovation in this area.

P0106URINE COMPLEMENT FRAGMENTS ARE ASSOCIATED WITH KIDNEY FUNCTION AND DISEASE ETIOLOGY

Abstract Background and Aims Changes in complement factors have been associated with chronic kidney disease, most prominently in the context of C3 glomerulopathy. Based on these reports, the hypothesis was generated that specific urine complement fragments are associated with kidney disease etiologies, and progression of the disease may be reflected by changes in these proteins or peptides. In this study, we aimed at investigating the occurrence of complement fragments in urine, and their association with kidney function in general and disease etiology in particular. Methods Urine samples from patients with kidney disease of different etiologies and control subjects with normal kidney function were investigated using tandem mass spectrometry coupled with capillary electrophoresis and liquid chromatography to identify complement derived peptides based on their amino acid sequence. Subsequently, distribution of these peptides in the different kidney disease etiologies and normal kidney function was investigated via analysis of datasets recorded in the human proteome/peptidome database. All datasets, where eGFR was available and at least one of the complement peptides detected, were included. Results Twenty one different urinary peptides derived from complement could be identified. These peptide fragments originated from the complement factors C3, C4A and C4B, and CFB. Further investigation of 4557 datasets recorded in the urine proteome/peptidome database meeting the aforementioned inclusion requirements (eg available eGFR values and with at least one of the complement peptides detected), revealed, for most C3-derived peptides, an inverse, while for the majority of peptides derived from CFB, a positive association with eGFR. The table lists the detected complement derived peptides, the parental protein (Complement X), rho and p-value of the association with eGFR, and the number of samples (n) where the peptide was detected. When investigating the urine complement fragments, as a result of disease etiology, disease-specific distribution was detected, indicating a distinct association of complement factors and associated proteases, depending on disease etiology. As an example, the peptide distribution in AKI, IgA-Nephropathy (IgA-N) and vasculitis is shown in the Figure. Conclusion Multiple complement-derived peptides are significantly associated with kidney function, and with certain kidney disease etiologies, such as AKI, IgA-N or vasculitis. Proteomic screening of these complement factors may provide a basis, not only for non-invasively assessing kidney disease, but also for an opportunity to develop novel drugs; the latter, via targeting the associated proteases responsible for the release of these complement-derived peptides, in a specific kidney disease manner, in urine.

Urine Proteomic Analysis Reveals Disease-Specific Patterns in Pediatric Patients with Classical Hodgkin's Disease(HD). an Addon Study to the Euronet-PHL-C2 Trial

Background HD is a B-lineage lymphoma characterized, depending on subtype, by a prominent inflammatory infiltrate and fibrosis. Clinically, inflammatory symptoms like fever, weight loss and night sweats (B-symptoms) and increased blood biomarkers of inflammation, including ESR and CRP, are characteristic of more advanced disease. The clinical trial EURONET-PHL-C2 (Second International Inter-Group Study for Classical Hodgkin's Lymphoma in Children and Adolescents) is a randomized, prospective trial that compares chemo- and radiotherapy treatment concepts of different intensities in patients with intermediate and advanced HD. Patients are stratified by risk into 3 therapy groups (TL-1 to TL-3). An ESR&gt; 30mm/h had been a risk factor for relapse in previous studies and leads to upstaging from the lowest (TL-1, Ann Arbor stage I and IIa without additional risk factors) to the intermediate risk group TL-2 in the current study. This addon pilot study tested urine proteomic patterns from pediatric patients with HD at diagnosis and compared them to the patterns of normal children. The questions were: Is there a HD-specific pattern, a pattern that identifies high risk or a pattern that correlates with inflammatory markers? Patients and methods Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to compare the peptide profiles in the mass range of 0.8 to 20 kDa of urine samples (N=34) from 16 children with pediatric Hodgkin lymphoma (PHL) as case and 32 age-matched children with no evidence of a disease (N=28) or with urinary tract infection (N=4) as control groups. Marker selection was based on a two-step strategy. First, a group-wise comparison of rank sum differences was performed on a set of 2418 annotated peptides with distribution frequencies above 30% in at least one of the groups with subsequent adjustment for multiple testing by the method of Bonferroni. In the second step marker candidates were further restricted to those demonstrating a significant positive or negative Spearman rho correlation coefficient (≥0.34 or ≤-0.34) to the Ann-Arbor classification criteria. From the resulting peptides a multivariate peptide marker classifier was established by support vector machine modeling and applied to an independent confirmation set of PHL (N=16, 31 urine samples) and control (N=18, 18 urine samples) patients to determine classification accuracy in receiver operating characteristics (ROC) analysis. Peptides included in the PHL classifier were resolved in their amino acid sequence by tandem mass spectrometry to identify the proteins from which the peptide markers are derived. Results The established multivariate peptide marker model consisting of 40 naturally occurring urinary peptides enabled absolute differentiation between PHL patients and children without signs of disease or urinary tract infection in independent validation as revealed by an area under the ROC curve value of 1.0 (95% confidence interval: 0.93 to 1.00, p&lt;0.0001). Amino acid sequencing revealed that the majority of peptides are interstitial collagen fragments from specific hot-spot regions within the proteins linear sequence and in part with overlapping amino acid sequences indicative for the activity of specific extracellular matrix degrading proteases. Other PHL peptide markers are derived from the tumor associated proteins S100-A9, Prostaglandin-H2 D-isomerase and Cytokeratin-8. Conclusions and perspective We were able to identify a proteomic pattern characteristic for HD, markers for relapse risk group or HD-associated inflammation were not yet identified. Disclosures Metzger: Mosaiques Diagnostics: Employment.

The use of tail-anchored protein chimeras to enhance liposomal cargo delivery.

BackgroundLiposomes are employed as drug delivery vehicles offering a beneficial pharmacokinetic/distribution mechanism for in vivo therapeutics. Therapeutic liposomes can be designed to target specific cell types through the display of epitope-specific targeting peptides on their surface. The majority of peptides are currently attached by chemical modification of lipid constituents. Here we investigate an alternative and novel method of decorating liposomes with targeting ligand, using remotely and spontaneously inserting chimeric tail-anchored membrane (TA) proteins to drug loaded liposomes.Methods and resultsAn artificial TA protein chimera containing the transmembrane domain from the spontaneously inserting TA protein cytochrome b5 (Cytb5) provided a robust membrane tether for the incorporation of three different targeting moieties into preformed liposomes. The moieties investigated were the transactivator of transcription (TAT) peptide, the EGF-receptor binding sequence GE11 and the placental and tumour homing ligand CCGKRK. In all cases, TA protein insertion neither significantly altered the size of the liposomes nor reduced drug loading. The efficacy of this novel targeted delivery system was investigated using two human cell lines, HeLa M and BeWo. Short term incubation with one ligand-modified TA chimera, incorporating the TAT peptide, significantly enhanced liposomal delivery of the encapsulated carboxyfluorescein reporter.ConclusionThe Cytb5 TA was successfully employed as a membrane anchor for the incorporation of the desired peptide ligands into a liposomal drug delivery system, with minimal loss of cargo during insertion. This approach therefore provides a viable alternative to chemical conjugation and its potential to accommodate a wider range of targeting ligands may provide an opportunity for enhancing drug delivery.

Gastric digestion of cow and goat milk: Peptides derived from simulated conditions of infant digestion.

Infant formula products are predominantly manufactured using cow milk protein; goat milk also provides a suitable protein source. In this study, we directly compared cow and goat milk protein digestion using pH and enzyme conditions to simulate infant gastric conditions. Generated peptides, identified using liquid chromatography coupled to a mass spectrometer, show both similarities and differences in cow and goat milk post-digestion profiles. The majority of peptides were from casein proteins, 50% representing β-casein, with many peptides unique to each species. Low or no peptides for β-Lactoglobulin and α-Lactalbumin, respectively, suggest these proteins were highly resistant to infant gastric digestion, as reported by others. Minor milk proteins, comprising 5% of peptides, were represented by different proteins from cow and goat. Peptides with known bioactivities were also observed, both in common and unique to each species. Together these data may explain reported differences in digestion characteristics of cow and goat milk.

Purification and identification of dipeptidyl peptidase IV and angiotensin-converting enzyme inhibitory peptides from silver carp (Hypophthalmichthys molitrix) muscle hydrolysate

Diabetes and hypertension are increasing threats to human health, which could be improved by inhibiting dipeptidyl peptidase IV (DPP-IV) and angiotensin-converting enzyme (ACE), respectively. Herein, we used five different enzymes to hydrolyze silver carp (Hypophthalmichthys molitrix) muscle protein. Among all treatments, the Neutrase-generated hydrolysate that was purified by a three-step isolation and then was identified using liquid chromatography–tandem mass spectrometry resulted in the most potent DPP-IV inhibitory activity and relatively high ACE inhibitory activity. The identified peptide Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg was the most effective at inhibiting DPP-IV with a half-maximal inhibitory concentration (IC50) value of 647.02 ± 4.30 µM. Leu-Leu-Asp-Leu-Gly-Val-Pro showed the highest ACE inhibitory activity with an IC50 value of 329.33 ± 15.53 µM. Lys-Ala-Val-Gly-Glu-Pro-Pro-Leu-Phe exhibited the dual inhibition against both DPP-IV and ACE with IC50 values of 1,317.39 ± 20.93 µM and 726.01 ± 8.69 µM, respectively. Interestingly, it seemed that the majority of peptides which were responsible for inhibiting DPP-IV were different from the ones which caused the inhibition of ACE.

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

ABSTRACTMutated proteins arising from somatic mutations in tumors are promising targets for cancer immunotherapy. They represent true tumor-specific antigens (TSAs) as they are exclusively expressed in tumors, reduce the risk of autoimmunity and are more likely to overcome tolerance compared to wild-type (wt) sequences. Hence, we designed a panel of long peptides (LPs, 28–35 aa) comprising driver gene mutations in TP35 and KRAS frequently found in gastrointestinal tumors to test their combined immunotherapeutic potential. We found increased numbers of T cells responsive against respective mutated and wt peptides in colorectal cancer patients that carry the tested mutations in their tumors than patients with other mutations. Further, active immunization of HLA(-A2/DR1)-humanized mice with mixes of the same mutated LPs yielded simultaneous, polyvalent CD8+/CD4+ T cell responses against the majority of peptides. Peptide-specific T cells possessed a multifunctional cytokine profile with CD4+ T cells showing a TH1-like phenotype. Two mutated peptides (Kras[G12V], p53[R248W]) induced significantly higher T cell responses than corresponding wt sequences and comprised HLA-A2/DR1-restricted mutated epitopes. However, vaccination with the same highly immunogenic LPs strongly increased systemic regulatory T cells (Treg) numbers in a syngeneic sarcoma model over-expressing these mutated protein variants and resulted in accelerated tumor outgrowth. In contrast, tumor outgrowth was delayed when vaccination was directed against tumor-intrinsic Kras/Tp53 mutations of lower immunogenicity. Conclusively, we show that LP vaccination targeting multiple mutated TSAs elicits polyvalent, multifunctional, and mutation-specific effector T cells capable of targeting tumors. However, the success of this therapeutic approach can be hampered by vaccination-induced, TSA-specific Tregs.

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.

Amino acid composition and antioxidative properties of hydrolysed pumpkin (Cucurbita pepo L.) oil cake protein

ABSTRACTPumpkin oil cake protein isolate (POCPI) was hydrolysed using two food-grade enzymes alcalase and trypsin. Alcalase-hydrolysed POCPI (POCPH1) was selected as the optimum treatment based on the DPPH radical scavenging, total antioxidative, and ferrous ion chelating activities. Amino acid composition showed a direct relationship between the antioxidative activity of POCPH1 and the amount of hydrophobic amino acids that formed 33.49% of its total amino acids. Surface hydrophobicity decreased as a result of hydrolysis and potent thermal and pH stability was observed in POCPH1 (p ˂ 0.05). In terms of molecular weight distribution, size exclusion chromatogram indicated that the majority of peptides possessed molecular weight less than 6.5 kDa. Based on the results, POCPH1 could be employed as a natural antioxidative agent with strong pH and thermal stability.

Modulation of Ion Channels by Cysteine-Rich Peptides: From Sequence to Structure.

Venom peptides are natural ligands of ion channels and have been used extensively in pharmacological characterization of various ion channels and receptors. In this chapter, we survey all known venom peptide ion-channel modulators. Our survey reveals that the majority of venom peptides characterized to date target voltage-gated sodium or potassium channels. We further find that the majority of these peptides are found in scorpion and spider venoms. We discuss the influence of the pharmacological tools available in biasing discovery and the classical "toxin-to-sequence" approach to venom peptide biodiscovery. The impact of high-throughput sequencing on the existing discovery framework is likely to be significant and we propose here an alternative "sequence-to-toxin" approach to peptide screening, relying more on recently developed high-throughput methods. Methods for production and characterization of disulfide rich toxins in a high-throughput setting are then described, focusing on bacterial protein expression and solution state structural characterization by NMR spectroscopy. Finally, the role of X-ray crystallography and cryo-EM are highlighted by discussing the currently known channel-peptide complexes.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Effects of Traveling Wave Ion Mobility Separation on Data Independent Acquisition in Proteomics Studies

qTOF mass spectrometry and traveling wave ion mobility separation (TWIMS) hybrid instruments (q-TWIMS-TOF) have recently become commercially available. Ion mobility separation allows an additional dimension of precursor separation inside the instrument, without incurring an increase in instrument time. We comprehensively investigated the effects of TWIMS on data-independent acquisition on a Synapt G2 instrument. We observed that if fragmentation is performed post TWIMS, more accurate assignment of fragment ions to precursors is possible in data independent acquisition. This allows up to 60% higher proteome coverage and higher confidence of protein and peptide identifications. Moreover, the majority of peptides and proteins identified upon application of TWIMS span the lower intensity range of the proteome. It has also been demonstrated in several studies that employing IMS results in higher peak capacity of separation and consequently more accurate and precise quantitation of lower intensity precursor ions. We observe that employing TWIMS results in an attenuation of the detected ion current. We postulate that this effect is binary; sensitivity is reduced due to ion scattering during transfer into a high pressure "IMS zone", sensitivity is reduced due to the saturation of detector digitizer as a result of the IMS concentration effect. This latter effect limits the useful linear range of quantitation, compromising quantitation accuracy of high intensity peptides. We demonstrate that the signal loss from detector saturation and transmission loss can be deconvoluted by investigation of the peptide isotopic envelope. We discuss the origin and extent of signal loss and suggest methods to minimize these effects on q-TWIMS-TOF instrument in the light of different experimental designs and other IMS/MS platforms described previously.

Abstract SY27-03: Biomarker-guided development of novel multi-peptide cancer vaccines - from discovery to phase lll trials.

Abstract SY27-03: Biomarker-guided development of novel multi-peptide cancer vaccines - from discovery to phase lll trials.