Mixture Of Synthetic Peptides Research Articles

Gradient Retention Factor Concept Applied to Method Development for Peptide Analysis by Means of RP-HPLC.

Using the van Deemter model, the efficiency of three stationary phase systems in the analysis of a mixture of synthetic peptides was evaluated: (i) monolithic, (ii) packed, and (iii) core-shell columns, and it was shown that the efficiency of the monolithic column is superior to the others, specifically using it, the lowest values of H min (0.03 and 0.1 mm) were obtained, and additionally its efficiency was not significantly affected by increasing the flow. Using the concept of the gradient retention factor (k*), a method for chromatographic separation of a peptide complex mixture was designed, implemented, and optimized and then transferred from a packed column to a monolithic one. The results showed that it was possible to separate all components of the mixture using both evaluated columns; moreover, the analysis time was reduced from 70 to 10 min, conserving the critical pair resolution (1.4), by the transfer method using the k* concept. The method developed was tested against a mixture of doping peptides, showing that this method is efficient for separating peptides of various natures. This investigation is very useful for the development of methods for the analysis of complex peptide mixtures since it provides a systematic approach that can be extrapolated to different types of columns and instrumentation.

Clustering and cross-linking of the wheat storage protein α-gliadin: A combined experimental and theoretical approach

Our aim was to understand mechanisms for clustering and cross-linking of gliadins, a wheat seed storage protein type, monomeric in native state, but incorporated in network while processed. The mechanisms were studied utilizing spectroscopy and high-performance liquid chromatography on a gliadin-rich fraction, in vitro produced α-gliadins, and synthetic gliadin peptides, and by coarse-grained modelling, Monte Carlo simulations and prediction algorithms. In solution, gliadins with α-helix structures (dip at 205 nm in CD) were primarily present as monomeric molecules and clusters of gliadins (peaks at 650- and 700-s on SE-HPLC). At drying, large polymers (Rg 90.3 nm by DLS) were formed and β-sheets increased (14% by FTIR). Trained algorithms predicted aggregation areas at amino acids 115–140, 150–179, and 250–268, and induction of liquid-liquid phase separation at P- and Poly-Q-sequences (Score = 1). Simulations showed that gliadins formed polymers by tail-to-tail or a hydrophobic core (Kratky plots and Ree = 35 and 60 for C- and N-terminal). Thus, the N-terminal formed clusters while the C-terminal formed aggregates by disulphide and lanthionine bonds, with favoured hydrophobic clustering of similar/exact peptide sections (synthetic peptide mixtures on SE-HPLC). Mechanisms of clustering and cross-linking of the gliadins presented here, contribute ability to tailor processing results, using these proteins.

Affinity Selection from Synthetic Peptide Libraries Enabled by De Novo MS/MS Sequencing

Recently, de novo MS/MS peptide sequencing has enabled the application of affinity selections to synthetic peptide mixtures that approach the diversity of phage libraries (> 108 random peptides). In conjunction with ‘split-mix’ solid phase synthesis to access equimolar peptide mixtures, this approach provides a straightforward means to examine synthetic peptide libraries of considerably higher diversity than has been feasible historically. Here, we offer a critical perspective on this work, report emerging data, and highlight opportunities for further methods refinement. With continued development, ‘affinity selection–mass spectrometry’ may become a complimentary approach to phage display, in vitro selection, and DNA-encoded libraries for the discovery of synthetic ligands that modulate protein function.

Characterization of HLA-A*02:01 MHC Immunopeptide Antigens Enhanced by Ultraviolet Photodissociation Mass Spectrometry.

Identifying major histocompatibility complex (MHC) class I immunopeptide antigens represents a key step in the development of immune-based targeted therapeutics and vaccines. However, the complete characterization of these antigens by tandem mass spectrometry remains challenging due to their short sequence length, high degree of hydrophobicity, and/or lack of sufficiently basic amino acids. This study seeks to address the potential for 193 nm ultraviolet photodissociation (UVPD) to improve the analysis of MHC class I immunopeptides by offering enhanced characterization of these sequences in lower charge states and differentiation of prominent isomeric leucine and isoleucine residues in the HLA-A*02:01 motif. Although electron transfer dissociation-higher energy collisional dissociation (EThcD) offered some success in the differentiation of leucine and isoleucine, 193 nm UVPD was able to confirm the identity of nearly 60% of leucine and isoleucine residues in a synthetic peptide mixture. Furthermore, 193 nm UVPD led to significantly more peptide identifications and higher scoring metrics than EThcD for peptides obtained from immunoprecipitation of MHC class I immunopeptides from in vitro cell culture. Additionally, 193 nm UVPD represents a promising complementary technique to higher-energy collisional dissociation (HCD), in which 424 of the 2593 peptides identified by 193 nm UVPD were not identified by HCD in HLA-A*02:01-specific immunoprecipitation and 804 of the 3300 peptides identified by 193 nm UVPD were not identified by HCD for pan HLA-A, -B, and -C immunoprecipitation. These results highlight that 193 nm UVPD offers an option for the characterization of immunopeptides, including differentiation of leucine and isoleucine residues.

Chip-Based Capillary Zone Electrophoresis Mass Spectrometry for Rapid Resolution and Quantitation of Critical Quality Attributes in Protein Biotherapeutics.

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

An 18O-Labeling Assisted LC-MS Method for Accurate Quantitation of Unprocessed C-Terminal Lysine in Therapeutic Monoclonal Antibodies.

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

T-cells modified with CD70 as an alternative cellular vaccine for antitumor immunity

Abstract Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T-cells. Dendritic cells (DCs) have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, antigenic peptide-loaded T-cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses, and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). T-cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T-cells but not CD4 T-cells or NK cells. The combination with PD-L1 blockade led to potent therapeutic efficacy which exhibited increased tumor infiltrating CD8 T-cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T-cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T-cells into tumor sites, and elicits enhanced therapeutic effects with peptide-based booster immunization. These results imply that T-cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

T Cells Modified with CD70 as an Alternative Cellular Vaccine for Antitumor Immunity.

PurposeSuccessful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T cells. Dendritic cells (DCs) are well-known potent antigen-presenting cells and have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, we aimed to develop T cell–based vaccine capable of eliciting potent antitumor therapeutic effects by providing effective costimulatory signals.Materials and MethodsAntigenic peptide-loaded T cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax).ResultsT cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T cells but not CD4 T cells or natural killer cells. The combination with programmed death-ligand 1 blockade led to potent therapeutic efficacy which exhibited increased tumor-infiltrating CD8 T cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T cells into tumor sites and elicits enhanced therapeutic effects with peptide-based booster immu-nization.ConclusionThese results imply that T cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

PATH-67. RECOMBINANT INTERLEUKIN-7 ENHANCES THE ANTI-TUMOR IMMUNITY OF A DENDRITIC CELL VACCINE IN A MURINE MALIGNANT GLIOMA

Abstract BACKGROUND Dendritic cell (DC)-based vaccines have been suggested as one of the promising immunotherapies for treating various cancers, including glioblastoma. We already developed a novel vaccination protocol with peptide-loaded DCs followed by a mixture of synthetic peptides, polyinosine- polycytidylic acid (poly-IC) and anti-CD40 antibodies (Trivax) in a melanoma mouse model. However, in a glioma mouse model, therapeutic efficacy is not as much as enough maybe due to relatively low antigenicity and blood brain barrier. MATERIAL AND METHODS IL-7, which is one of the most important cytokines to expand and develop T cells with anti-tumor immunity, was co-administrated intravenously with Trivax in an orthotopic murine malignant glioma. RESULTS Co-administration of the Trivax and recombinant IL-7 (Trivax7) increased the number of survivin specific T cells measured using ELISPOT assay and the population of central memory T cells, comparing with administration of Trivax. The tumor size of orthotopic mouse model in Trivax 7 group was smaller than those of Trivax only group. Finally, overall survival in Trivax 7 was longer than those of Trivax only. In addition, there was a prolonged survival of antigen-specific T cells in Trivax7 group than Trivax only group. CONCLUSION In summary, our novel combinational immunotherapy may overcome the limitations of current cell-based cancer vaccines and could be applicable for the treatment of glioblastoma patients.

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*[S

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.

Universal retention standard for peptide separations using various modes of high-performance liquid chromatography

Peptide retention standards are widely used by chromatography specialists. They can be used for quality control of peptide separations (separation efficiency, selectivity, retention values) and for accurate concatenation of retention data from multiple acquisitions in proteomics. So far the repertoire of available retention standards is mostly limited to reversed-phase separations. We introduce a synthetic peptide mixture which can be used in conjunction with the most popular peptide separation techniques: reversed-phase (RPLC), strong-cation exchange (SCX), (strong-anion exchange) SAX and hydrophilic interaction liquid chromatography (HILIC). Target sequences were first designed in-silico using Sequence-Specific Retention Calculator models covering all major peptide separation mechanisms. Peptides were also designed while keeping in mind the simplicity of retention time assignment using MS detection: they all have nearly identical masses and identical intense y3 fragment ions. This contribution demonstrates the application of this mixture for characterization of eight HILIC as well as SAX, SCX and C18 columns.

Role of P-53 Gene in Preventing Breast Cancer

Breast cancer affects more than one million patients annually in the world and is a leading cause of mortality. Histological type, grade, tumor size, lymph node involvement, and estrogen receptor and HER-2 receptor status, all influence prognosis and the probability of response to systemic therapies. P53 gene is mutated in about 30% of breast cancers/.The possible links between alterations of p53 and clinical or pathological features of breast tumors have been widely investigated. The first study to examine gene-expression patterns of breast cancer suggested that at least four major molecular classes of breast cancer exist: luminal-like, basal-like, normal like, and HER-2 positive. Basal-like breast cancer account for 15% of breast cancers and are often described as triple negative breast cancers (TNBCs). In fact, TNBCs, defined by lack of expression of estrogen receptor, progesterone receptor, and HER2, probably include both basal-like breast cancers and some poorly differentiated luminal breast cancers. They are also associated with a younger age and a poor prognosis. TNBCs also have an increased frequency of TP53 mutations. Recently, it was shown that mutant p53 status was a strongly unfavorable prognostic factor for relapse-free survival and overall survival only in a triple negative group in patients treated with adjuvant anthracycline-containing chemotherapy. Inflammatory breast cancer (IBC) is a clinical diagnosis known as the T4d category in the TNM classification. It is a distinct clinical subtype of locally advanced breast cancer (LABC), with a particularly aggressive behavior and poor prognosis. TP53 mutations are more frequent in inflammatory breast cancer (50%) than in non-inflammatory breast cancer (20–30%). The results from these studies served as the justification for attempts to vaccinate patients using p53-derived peptides, and a number of clinical trials are in progress. The most advanced work used a long synthetic peptide mixture derived from p53 (p53-SLP; Netherlands).The vaccine is delivered in the adjuvant setting and induces T helper type I response in the majority of patients. However, the response may not be potent enough to result in clinical benefit as a mono-therapy because most patients had T-helper cells that failed to produce key cytokines, indicating that this treatment should also be associated with another type of conventional or immunotherapy therapy.

Parsing and Quantification of Raw Orbitrap Mass Spectrometer Data Using RawQuant.

Effective analysis of protein samples by mass spectrometry (MS) requires careful selection and optimization of a range of experimental parameters. As the output from the primary detection device, the "raw" MS data file can be used to gauge the success of a given sample analysis. However, the closed-source nature of the standard raw MS file can complicate effective parsing of the data contained within. To ease and increase the range of analyses possible, the RawQuant tool was developed to enable parsing of raw MS files derived from Thermo Orbitrap instruments to yield meta and scan data in an openly readable text format. RawQuant can be commanded to export user-friendly files containing MS1, MS2, and MS3 metadata as well as matrices of quantification values based on isobaric tagging approaches. In this study, the utility of RawQuant is demonstrated in several scenarios: (1) reanalysis of shotgun proteomics data for the identification of the human proteome, (2) reanalysis of experiments utilizing isobaric tagging for whole-proteome quantification, and (3) analysis of a novel bacterial proteome and synthetic peptide mixture for assessing quantification accuracy when using isobaric tags. Together, these analyses successfully demonstrate RawQuant for the efficient parsing and quantification of data from raw Thermo Orbitrap MS files acquired in a range of common proteomics experiments. In addition, the individual analyses using RawQuant highlights parametric considerations in the different experimental sets and suggests targetable areas to improve depth of coverage in identification-focused studies and quantification accuracy when using isobaric tags.

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Evaluating the Characteristics of Reporter Ion Signal Acquired in the Orbitrap Analyzer for Isobaric Mass Tag Proteome Quantification Experiments.

Multiplexed quantification with isobaric chemical tags (e.g., TMT, iTRAQ) provides a robust and efficient means to comparatively examine proteome dynamics between several biological states using a mass spectrometer (MS). The quantitative nature of isobaric tags necessitates strict validation of the observed ion signals in the chosen MS detector before differential patterns are extracted between biological states. We present an in-depth analysis of isobaric tag data acquired on current generation Orbitrap MS hardware to illustrate pitfalls in acquisition settings that can negatively impact results. We establish, for the first time, the presence of a notch, a region of no observed values, in the reporter ion distributions from isobaric-labeled peptide mixtures acquired on these instruments. We determine that this notch is present in published data across a wide range of instruments of the same or different type and is isolated to the Orbitrap mass analyzer. We demonstrate that the impact of the notch can be minimized using manipulations of Orbitrap scan parameters and on-column injection amounts. Lastly, using a mixture of synthetic standard peptides we investigated the impact on identification rates and quantification precision. Together, these data highlight an important phenomenon that negatively impacts peptide identification and quantification in the Orbitrap analyzer as well as outlining guidelines to follow to ensure minimization of MS-induced artifacts in isobaric tag experiments resulting from the notch.

Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry

Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) −electrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?

Coupling of motion of the ion clouds with close m/z values is a well-established phenomenon for ion- trapping mass analyzers. In Fourier transform ion cyclotron resonance mass spectrometry it is known as ion coalescence. Recently, ion coalescence was demonstrated and semiquantitatively characterized for the Orbitrap mass analyzer as well. When it occurs, the coalescence negatively affects the basic characteristics of a mass analyzer. Specifically, the dynamic range available for the high resolving power mass measurements reduces. In shotgun proteomics, another potentially adverse effect of ion coalescence is interference of the isotopic envelopes for the coeluting precursor ions of close m/z values, subjected to isolation before fragmentation. In this work we characterize coalescence events for synthetic peptide mixtures with fully and partially overlapping (13)C-isotope envelopes including pairs of peptides with glutamine deamidation. Furthermore, we demonstrate that fragmentation of the otherwise coalesced peptide ion clouds may remove the locking between them owing to the total charge redistribution between more ion species in the mass spectrum. Finally, we estimated the possible scale of the coalescence phenomenon for shotgun proteomics by considering the fraction of coeluted peptide pairs with the close masses using literature data for the yeast proteome. It was found that up to one tenth of all peptide identifications with the relative mass differences of 20 ppm or less in the corresponding pairs may potentially experience the coalescence of the (13)C-isotopic envelopes. However, sample complexity in a real proteomics experiment and precursor ion signal splitting between many channels in tandem mass spectrometry drastically increase the threshold for coalescence, thus leading to practically coalescence-free proteomics based on Fourier transform mass spectrometry.

An optimized peptide vaccine strategy capable of inducing multivalent CD8+ T cell responses with potent antitumor effects

Therapeutic cancer vaccines are an attractive alternative to conventional therapies for treating malignant tumors, and successful tumor eradication depends primarily on obtaining high numbers of long-lasting tumor-reactive CD8+ T cells. Dendritic cell (DC)-based vaccines constitute a promising approach for treating cancer, but in most instances low immune responses and suboptimal therapeutic effects are achieved indicating that further optimization is required. We describe here a novel vaccination strategy with peptide-loaded DCs followed by a mixture of synthetic peptides, polyinosine-polycytidylic acid (poly-IC) and anti-CD40 antibodies (TriVax) for improving the immunogenicity and therapeutic efficacy of DC-based vaccines in a melanoma mouse model. TriVax immunization 7–12 d after priming with antigen-loaded DCs generated large numbers of long-lasting multiple antigen-specific CD8+ T cells capable of recognizing tumor cells. These responses were far superior to those generated by homologous immunizations with either TriVax or DCs. CD8+ T cells but not CD4+ T cells or NK cells mediated the therapeutic efficacy of this heterologous prime-boost strategy. Moreover, combinations of this vaccination regimen with programmed cell death-1 (PD-1) blockade or IL2 anti-IL2 antibody complexes led to complete disease eradication and survival enhancement in melanoma-bearing mice. The overall results suggest that similar strategies would be applicable for the design of effective therapeutic vaccination for treating viral diseases and various cancers, which may circumvent current limitations of cell-based cancer vaccines.

Laser desorption ionization mass spectrometry of peptides on a hybrid CHCA organic-inorganic matrix.

We report applications of new hybrid organic-inorganic silica based materials as laser desorption/ionization (LDI)-promoting surfaces for high-throughput identification of peptides. The driving force of our work was to design a new material composed of a conventional MALDI matrix covalently attached to silica with a high organic/inorganic ratio in order to improve the UV absorption by such LDI hybrid matrices. Amorphous CHCA-functionalized silica presenting an organic content up to 1.3 mmol g(-1) (around 40% in weight from TGA and elementary analysis measurements) gave very interesting LDI performances in terms of detection sensitivity as well as relative ionization discrepancy (spectral suppression) through the analyses of small synthetic peptide mixtures (550-1300 Da) taking CHCA and amorphous silica as model matrices for control experiments.

Proteome Informatics Research Group (iPRG)_2012: A Study on Detecting Modified Peptides in a Complex Mixture

The proteome informatics research group of the Association of Biomolecular Resource Facilities conducted a study to assess the community's ability to detect and characterize peptides bearing a range of biologically occurring post-translational modifications when present in a complex peptide background. A data set derived from a mixture of synthetic peptides with biologically occurring modifications combined with a yeast whole cell lysate as background was distributed to a large group of researchers and their results were collectively analyzed. The results from the twenty-four participants, who represented a broad spectrum of experience levels with this type of data analysis, produced several important observations. First, there is significantly more variability in the ability to assess whether a results is significant than there is to determine the correct answer. Second, labile post-translational modifications, particularly tyrosine sulfation, present a challenge for most researchers. Finally, for modification site localization there are many tools being employed, but researchers are currently unsure of the reliability of the results these programs are producing.