Peptide Elution Research Articles

Characterization of hybrid organo-silica monoliths for possible application in the gradient elution of peptides.

We characterized thermally polymerized organo-silica hybrid monolithic capillaries to test their applicability in the gradient elution of peptides. We have used a single-pot approach utilizing 3-(methacryloyloxy)propyltrimethoxysilane (MPTMS), ethylene dimethacrylate (EDMA), and n-octadecyl methacrylate (ODM) as functional monomers. The organo-silica monolith containing MPTMS and EDMA was compared with the stationary phase prepared by adding ODM to the original polymerization mixture. Column prepared using a three-monomer system provided a lower accessible volume of flow-through pores, a higher proportion of mesopores, and higher efficiency. We utilized isocratic and gradient elution data to predict peak widths in gradient elution. Both protocols provided comparable results and can be used for peptide peak width prediction. However, applying gradient elution data for peak width prediction seems simpler. Finally, we tested the effect of gradient time on achievable peak capacity in the gradient elution of peptides with a column prepared with a three-monomer system providing a higher peak capacity. However, the performance of hybrid organo-silica monolithic stationary phases in gradient elution of peptides must be improved compared to other monolithic stationary phases. The limiting factor is column efficiency in highly aqueous mobile phases, which needs to be focused on.

MassSpecPreppy-An end-to-end solution for automated protein concentration determination and flexible sample digestion for proteomics applications.

In proteomics, fast, efficient, and highly reproducible sample preparation is of utmost importance, particularly in view of fast scanning mass spectrometers enabling analyses of large sample series. To address this need, we have developed the web application MassSpecPreppy that operates on the open science OT-2 liquid handling robot from Opentrons. This platform can prepare up to 96 samples at once, performing tasks like BCA protein concentration determination, sample digestion with normalization, reduction/alkylation and peptide elution into vials or loading specified peptide amounts onto Evotips in an automated and flexible manner. The performance of the developed workflows using MassSpecPreppy was compared with standard manual sample preparation workflows. The BCA assay experiments revealed an average recovery of 101.3% (SD:±7.82%) for the MassSpecPreppy workflow, while the manual workflow had a recovery of 96.3% (SD:±9.73%). The species mix used in the evaluation experiments showed that 94.5% of protein groups for OT-2 digestion and 95% for manual digestion passed the significance thresholds with comparable peptide level coefficient of variations. These results demonstrate that MassSpecPreppy is a versatile and scalable platform for automated sample preparation, producing injection-ready samples for proteomics research.

Factors Influencing the Prediction Accuracy of Model Peptides in Reversed-Phase Liquid Chromatography

Hydrophobicity is an important physicochemical property of peptides in solution. As well as being strongly associated with peptide stability and aggregation, hydrophobicity governs the solution based chromatographic separation processes, specifically reversed-phase liquid chromatography (RPLC). In addition, hydrophobicity is a major physicochemical property of peptides in comparison to H-bonding, electrostatic, and aromatic properties in intermolecular interactions. However, a wide range of molecular factors can influence peptide hydrophobicity, with accurate predictions depending on specific peptide amino acid compositions, structure, and conformation. It is noticeable that peptide composition, the position of the amino acid, and its neighbouring groups play a crucial role in the elution process. In light of this, the same amino acid behaved differently depending on its position and neighbouring amino acid in the peptide chain. Extra attention should be paid to the denaturation process during the course of elution, as it has been shown to complicate and alter the elution pattern. This paper reports on the key peptide properties that can alter hydrophobicity and, consequently, the RPLC elution behaviour of the peptides, and it will conclude by proposing improved prediction algorithms for peptide elution in RPLC.

Cancer-specific T helper shared and neo-epitopes uncovered by expression of the MHC class II master regulator CIITA.

We report an approach to identify tumor-specific CD4+ Tcell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral- and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ Tcell responses, which helped tumor control invivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important targets for cancer immunotherapy.

Abstract 1379: Discovery of tumor-associated, immunogenic peptides presented in a patient-derived, mutant calreticulin-driven myeloproliferative neoplasm cell line

Abstract Background: Myeloproliferative neoplasms (MPNs) are a type of chronic blood cancers. A subset are driven by frameshift mutations in the calreticulin gene (CALR). The CALR protein is a lectin chaperone and primarily assists in glycoprotein folding in the endoplasmic reticulum. It is also part of the peptide loading complex, a set of proteins involved in antigen processing and peptide presentation by major histocompatibility complex class I (MHC-I) molecules. All MPN-associated frameshift mutations in CALR lead to a similar change in its C-terminal, altering its protein-protein interactions. Examples include impaired chaperone activity of mutant CALR that impacts protein homeostasis and compromised activity of the MHC-I peptide loading complex. We hypothesized that these impaired functions will alter the repertoire of peptides presented by MHC-I in mutant CALR expressing cells, which we characterized in a patient-derived, mutant-CALR-driven myeloid tumor cell line, Marimo. Methods: We carried out an immunopeptidome analysis on Marimo cell line derivates expressing either wildtype or mutant CALR to identify potential MPN-associated peptides presented by MHC-I, and validated their immunogenicity in human samples. MHC-I-peptide complexes were immunoprecipitated from these Marimo cell line derivates, followed by peptide elution and analysis by mass spectrometry. A subset of peptides that were found in the mass spectrometry analysis of mutant CALR cells but not wildtype CALR cells were tested for their immunogenicity by assaying the in vitro T cell responses they evoked. Naïve T cells from healthy donors, carrying at least one MHC-I allele that matched the Marimo cells, were primed and expanded with the selected peptides and peptide-specific T cell responses were measured by intracellular staining detecting effector cytokines IFN-γ and TNF-α. Results: 1. The expression of mutant CALR disrupts the function of the peptide loading complex, leading to reduced recruitment of MHC-I and reducing its cell surface expression by almost 40% compared to cells expressing wildtype CALR. 2. Peptide repertoire of MHC-I is significantly altered by the expression of mutant CALR. Immunopeptidomics revealed a qualitative and quantitative difference in peptides presented by cells expressing wildtype CALR vs mutant CALR 3. The immunogenicity of a curated list of 24 peptides was tested in healthy donors, and at least 6 peptides activated T cell responses. Similar testing in MPN patients is planned. Conclusions: This is the first study to investigate the impact of mutant CALR on the repertoire of peptides presented by MHC-I in myeloid cells. We discovered tumor-associated, immunogenic peptides that may serve as potential targets of immunotherapy allowing the specific elimination of mutant CALR-expressing cells, which has been a challenge in the treatment of MPNs. Citation Format: Najla Arshad, Nathalie Vigneron, Cansu Cimen Bozkus, Vincent Stroobant, Stefan Naulaerts, Nina Bhardwaj, Benoît Van den Eynde, Peter Cresswell. Discovery of tumor-associated, immunogenic peptides presented in a patient-derived, mutant calreticulin-driven myeloproliferative neoplasm cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1379.

NAPT, an unbiased approach for sequential analysis of the protein N- and C-terminome.

Protein termini are generally associated with protein stability. Taking advantage of charge state differences between terminal peptides and internal peptides, we developed a new method termed LysargiNase-assisted Analysis of the Protein N- and C-Terminome (NAPT) for selective separation of protein termini. Altogether, we identified 2458 protein N-termini and 3056 protein C-termini from the HeLa cell line.

Microfluidic platform based on site-specific post-imprinting modification of molecularly imprinted monolith with Connizzaro reaction to improve identification of N-myristoylated peptides

In order to improve the identification of N-myristoylated peptides by mass spectrometry, an enrichment strategy based on microfluidic platform was presented by combining molecularly imprinted polymer (MIPs) with post-imprinting modification and trypsin-immobilized enzyme reactor (IMER). 4-Allyloxybenzaldehyde was selected as covalent functional monomer to synthesize schiff base with the terminal amino group of the template, N-myristoylated-glycine-serine-asparagine-lysine (Myr-GSNK). With N, N′-methylenebisacrylamide as functional monomer, N, N′-methylenebisacrylamide was selected as crosslinking monomer, PSS-octavinyl substituted as co-crosslinker, and DMSO and dodecanol as porogen to prepare Myr-GSNK MIP monolith. After the Schiff base was cleaved, along with the elution of the template peptides, the post-imprinting treatment was conducted to transform the residues of aldehyde to hydroxyl and carboxyl groups. The resulting imprinted monolith was used as an apparatus for peptides enrichment in a microfluidic platform composed of a chip and IMER for treatment of the proteins extracted from MCF-7 cells. A total of 1296 proteins and 5670 peptides can be identified, including 71 myristoylated proteins and 78 myristoylated peptides. Compared with the previous enrichment material against myristoylated peptides, the MIP monolith showed greater selectivity and adsorption capacity, which provided an efficient means for the protein identification of myristoylated proteomes in complex biological samples.

RIPPA: Identification of MHC-II Binding Peptides from Antigen Using a Yeast Display-Based Approach.

Mapping MHC-II binding peptides derived from an antigenic protein for potential CD4+ T-cell epitopes has been challenging due to a lack of experimental approaches that are both quantitative and rapid. The rate-limiting steps in current approaches include the construction of single MHC allele expressing cell lines and/or the purification of the MHC-II allelic proteins for peptide elution (i.e., mass spectrometry) or in vitro peptide binding (i.e., ELISA) assays. These labor-intensive steps typically take up to 4 months or more. In this protocol, we describe a system that uses yeast cells to display "empty" (i.e., without covalently linked peptides) MHC-II heterodimers that are capable of binding exogenously added peptides of interest. This yeast-MHC-II system eliminates the time-consuming soluble MHC-II purification steps, allowing rapid identification of peptide ligands from protein antigens (RIPPA). The amount of peptide loading to MHC-II or the extent of competition between indicator and competitor peptides at the surface of yeast cells can be quantitatively determined using flow cytometric analysis. Importantly, the protocol only takes ∼1 month from the construction of plasmids and the yeast display of "empty" MHC-II to the quantitative determination of MHC-II binding peptides from a given antigen. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Yeast display of "empty" MHC-II Support Protocol: Construction of yeast shuttle vector expressing "empty" MHC-II Basic Protocol 2: Peptide competition on the surface of yeast cells Alternate Protocol: RIPPA in a 96-well format.

Citrullinated Epitopes Identified on Tumour MHC Class II by Peptide Elution Stimulate Both Regulatory and Th1 Responses and Require Careful Selection for Optimal Anti-Tumour Responses

BackgroundSomatic mutations or post-translational modifications of proteins result in changes that enable immune recognition. One such post-translational modification is citrullination, the conversion of arginine residues to citrulline. Citrullinated peptides are presented on MHC class II (MHCII) via autophagy which is upregulated by cellular stresses such as tumourigenesis.MethodsPeptides were eluted from B16 melanoma expressing HLA-DP4 and analysed by mass spectrometry to profile the presented citrullinated repertoire. Initially, seven of the identified citrullinated peptides were used in combination to vaccinate HLA-DP4 transgenic mice. Immune responses were characterised from the combination and individual vaccines by ex vivo cytokine ELISpot assay and assessed for tumour therapy.ResultsThe combination vaccine induced only weak anti-tumour therapy in the B16cDP4 melanoma model. Immune phenotyping revealed a dominant IFNγ response to citrullinated matrix metalloproteinase-21 peptide (citMMP21) and an IL-10 response to cytochrome p450 peptide (citCp450). Exclusion of the IL-10 inducing citCp450 peptide from the combined vaccine failed to recover a strong anti-tumour response. Single peptide immunisation confirmed the IFNγ response from citMMP21 and the IL-10 response from citCp450 but also showed that citrullinated Glutamate receptor ionotropic (citGRI) peptide stimulated a low avidity IFNγ response. Interestingly, both citMMP21 and citGRI peptides individually, stimulated strong anti-tumour responses that were significantly better than the combined vaccine. In line with the citGRI T cell avidity, it required high dose immunisation to induce an anti-tumour response. This suggests that as the peptides within the combined vaccine had similar binding affinities to MHC-II the combination vaccine may have resulted in lower presentation of each epitope and weak anti-tumour immunity.ConclusionWe demonstrate that tumours present citrullinated peptides that can stimulate Th1 and regulatory responses and that competition likely exists between similar affinity peptides. Characterisation of responses from epitopes identified by peptide elution are necessary to optimise selection for tumour therapy.

Drug hapten-specific T-cell activation: Current status and unanswered questions.

Drug haptens are formed from the irreversible, covalent binding of drugs to nucleophilic moieties on proteins, which can warrant adverse reactions in the body including severe delayed-type, T-cell mediated, drug hypersensitivity reactions (DHRs). While three main pathways exist for the activation of T-cells in DHRs, namely the hapten model, the pharmacological interaction model and the altered peptide repertoire model, the exact antigenic determinants responsible have not yet been defined. In recent years, progress has been made using advanced mass spectrometry-based proteomic methods to identify protein carriers and characterise the structure of drug-haptenated proteins. Since genome-wide association studies discovered a link between human leukocyte antigens (HLA) and an individual's susceptibility to DHRs, much effort has been made to define the drug-associated HLA ligands driving T-cell activation, including the elution of natural HLA peptides from HLA molecules and the generation of HLA-binding peptides. In this review, we discuss our current methodology used to design and synthesise drug-modified HLA ligands to investigate their immunogenicity using T-cell models, and thus their implication in drug hypersensitivity.

Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens (RIPPA).

CD4+ T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes. To study these responses, it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition. However, generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming. Here, we express human MHC alleles (HLA-DR4 and HLA-DQ6) as native, noncovalent αβ dimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens. We demonstrate rapid, accurate identification of DQ6 ligands from pre-pro-hypocretin, a narcolepsy-related immunogenic target. We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes, and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4+ T cells from unexposed individuals carrying DR4 subtypes. Our method is optimized for immediate application upon the emergence of novel pathogens.

A cell-free antigen processing system reveals antigenic factors critical for HIV-1 epitope dominance and informs vaccine design

Abstract Certain CD4+T cell epitopes have been associated with immune control of HIV-1 replication and the presence of anti-Env neutralizing antibodies. However, it remains unclear what antigen-dependent factors influence the dominance of certain epitopes in HIV-1 proteins. To study HIV-1 epitope dominance, we used a cell-free antigen processing system previously developed by our group that utilizes soluble HLA-DR1*01:01 (DR1), HLA-DM (DM), protein antigen and three cathepsins in a reducing and low pH environment to allow for antigen binding and cleavage. We subjected HIV-1 antigens from Gag, Pol, Env and accessory proteins Vif, Nef, Tat, and Rev to this system. Immunoprecipitation of pMHC-II complexes followed by peptide elution and identification via LC-MS/MS revealed a map of DR1-bound epitopes across the near-full length HIV-1 proteome. Most identified epitopes were DM-resistant, and in some cases, DM narrowed the epitope diversity to one species. Importantly, we identified 28 novel epitopes, including several glycopeptides eluted from DR1. We noted epitope “hot spots” in HIV-1 antigens and striking similarities in epitopes from HIV polyproteins (Gag precursor) versus individual subunits (Gag capsid). Analysis of the location of several epitopes from smaller HIV-1 proteins based on their known crystal structures mapped to regions of low protein stability and higher solvent accessibility. Finally, we found that several epitopes induced memory CD4+T cell responses in persons living with HIV-1. Thus, by using a minimalist in vitro processing system, we can interrogate novel HIV-1 epitopes and understand how DM-resistance and antigen structure influence epitope dominance, which has important implications for vaccine design.

Total Retention Liquid Chromatography-Mass Spectrometry to Achieve Maximum Protein Sequence Coverage.

Peptide identification by liquid chromatography-mass spectrometry (LC-MS) requires retention and elution of peptides from the LC column. Although medium and hydrophobic peptides are readily retained by the C18 columns that are commonly used in proteomics, short and hydrophilic peptides are not retained nor measured by MS due to their elution in the void volume after sample injection. These nonretained peptides can possess important post-translational modifications, such as glycosylation or phosphorylation. We describe a total retention LC-MS method that employs a reverse phase C18 column and porous graphitic carbon (PGC) column to retain both hydrophobic and hydrophilic peptides for LC-MS analysis. Our setup uses a single valve with a trapping column and two LC pumps run at low microliter/minute flow rates to deliver separate gradients to parallel capillary C18 and PGC columns. Our capillary LC system balances the need for high sensitivity with ease of implementation as compared to other 2D LC systems that use nanocolumns with multiple trapping columns and multiport valves. We demonstrate the utility of the method identifying hydrophilic peptides that went undetected when only a C18 nanocolumn was used. These missed hydrophilic peptides include tripeptides and N-glycosylated species.

Adsorption of Amyloid Beta Peptide by Metal-Organic Frameworks.

Metal–organic frameworks (MOFs) are capable of adsorbing a wide range of molecules. In addition to the more commonly investigated small molecules, researchers have demonstrated that MOFs adsorb much larger molecules, such as proteins and peptides. We have investigated whether MOFs are capable of adsorbing amyloid beta peptide. Amyloid beta plays a pivotal role in the progression of Alzheimer’s disease because individual copies of the peptides can aggregate, forming neurotoxic oligomers and the amyloid plaques found in brains of Alzheimer’s patients. After synthesizing a number of commonly studied MOFs, their adsorption capabilities were tested. We found that the MOFs tested readily adsorbed small amounts of amyloid beta (as determined by gel electrophoresis). It was determined that in most cases, adsorption occurs rapidly, with complete adsorption within minutes of incubation. Overall adsorption capacity was found to vary between different MOFs as well. Once adsorbed, amyloid beta peptide can subsequently be eluted from some MOFs by treatment with acetonitrile/water solutions, though retention strength varied between different MOFs. In some cases, MOFs that showed complete adsorption also saw high levels of peptide elution, but others showed little to no elution of the peptide. Together these data can help us begin to understand the interactions between amyloid beta and MOFs.

What We See, What We Do Not See, and What We Do Not Want to See in HLA Class I Immunopeptidomes.

The identification of peptides bound to human leukocyte antigen class I (HLA-I) molecules-that is, the HLA-I immunopeptidome-is a useful tool in the hunt for epitopes suitable for vaccinations and immunotherapies. These peptides are mainly generated by proteasomes through peptide hydrolysis and peptide splicing. In this issue, Nicastri and colleagues compared different methods for the elution of HLA class I-associated peptides. It is demonstrated that the choice of HLA-associated peptide enrichment and purification strategy affects peptide yields and creates a bias in detected sequence repertoire. The author carried out this technical brief through the analysis of canonical non-spliced peptides. However, their study left out any analysis of post-translationally spliced peptides, thereby missing an opportunity to shed light on the persistent debate of the frequency of these unconventional peptides.

Implementation of a multiple-fraction concatenation strategy in an online two-dimensional high-/low-pH reversed-phase/reversed-phase liquid chromatography platform for qualitative and quantitative shotgun proteomic analyses.

Multidimensional liquid chromatography is the mainstay separation technique used for shotgun proteomic analyses. The application of a multiple-fraction concatenation (MFC) strategy can result in a more disperse and consistent peptide elution profile across different fractions, when compared with a conventional strategy. Herein, we present the first automated online RP-RP platform implementing an MFC strategy to facilitate robust, unattended, routine proteomic analyses. The improved duty cycle utilization of the MFC strategy led to an increase of 9% in the separation space occupancy and increases of approximately 10% in the identification of both proteins and peptides. The peptides uniquely identified by the MFC strategy were significantly biased toward those of acidic nature, with increased precursor signals leading to improved MS/MS spectral quality and enhanced acidic peptide identification. These improvements in qualitative analysis using the MFC strategy were also extended to quantitative analysis. When the acquired proteome was quantified with a normalized spectral abundance factor, the additionally acquired acidic peptides were a critical factor leading to enhanced reproducibility of quantitation using the MFC strategy. With merits of superior qualitative and quantitative characteristics over the conventional strategy, the MFC strategy appears to be a highly amenable technique for enhancing the separation capacity for routine proteomic analyses.

Uncovering the Tumor Antigen Landscape: What to Know about the Discovery Process.

According to the latest available data, cancer is the second leading cause of death, highlighting the need for novel cancer therapeutic approaches. In this context, immunotherapy is emerging as a reliable first-line treatment for many cancers, particularly metastatic melanoma. Indeed, cancer immunotherapy has attracted great interest following the recent clinical approval of antibodies targeting immune checkpoint molecules, such as PD-1, PD-L1, and CTLA-4, that release the brakes of the immune system, thus reviving a field otherwise poorly explored. Cancer immunotherapy mainly relies on the generation and stimulation of cytotoxic CD8 T lymphocytes (CTLs) within the tumor microenvironment (TME), priming T cells and establishing efficient and durable anti-tumor immunity. Therefore, there is a clear need to define and identify immunogenic T cell epitopes to use in therapeutic cancer vaccines. Naturally presented antigens in the human leucocyte antigen-1 (HLA-I) complex on the tumor surface are the main protagonists in evocating a specific anti-tumor CD8+ T cell response. However, the methodologies for their identification have been a major bottleneck for their reliable characterization. Consequently, the field of antigen discovery has yet to improve. The current review is intended to define what are today known as tumor antigens, with a main focus on CTL antigenic peptides. We also review the techniques developed and employed to date for antigen discovery, exploring both the direct elution of HLA-I peptides and the in silico prediction of epitopes. Finally, the last part of the review analyses the future challenges and direction of the antigen discovery field.

Immunopeptidome Analysis of HLA-DPB1 Allelic Variants Reveals New Functional Hierarchies.

HLA-DP alleles can be classified into functional T cell epitope (TCE) groups. TCE-1 and TCE-2 are clearly defined, but TCE-3 still represents an heterogeneous group. Because polymorphisms in HLA-DP influence the presented peptidome, we investigated whether the composition of peptides binding in HLA-DP may be used to refine the HLA-DP group classification. Peptidomes of human HLA-DP-typed B cell lines were analyzed with mass spectrometry after immunoaffinity chromatography and peptide elution. Gibbs clustering was performed to identify motifs of binding peptides. HLA-DP peptide-binding motifs showed a clear association with the HLA-DP allele-specific sequences of the binding groove. Hierarchical clustering of HLA-DP immunopeptidomes was performed to investigate the similarities and differences in peptidomes of different HLA-DP molecules, and this clustering resulted in the categorization of HLA-DP alleles into 3-DP peptidome clusters (DPC). The peptidomes of HLA-DPB1*09:01, -10:01, and -17:01 (TCE-1 alleles) and HLA-DPB1*04:01, -04:02, and -02:01 (TCE-3 alleles) were separated in two maximal distinct clusters, DPC-1 and DPC-3, respectively, reflecting their previous TCE classification. HLA-DP alleles categorized in DPC-2 shared certain similar peptide-binding motifs with DPC-1 or DPC-3 alleles, but significant differences were observed for other positions. Within DPC-2, divergence between the alleles was observed based on the preference for different peptide residues at position 9. In summary, immunopeptidome analysis was used to unravel functional hierarchies among HLA-DP alleles, providing new molecular insights into HLA-DP classification.

Highly sensitive detection of phosphopeptides with superparamagnetic Fe3O4@mZrO2 core-shell microspheres-assisted mass spectrometry

Protein phosphorylation is one of the most important post-translational modifications. It is an active research area to study phosphoproteomics for discovery of disease biomarkers and druggable targets. Here we report the development of superparamagnetic Fe3O4@mZrO2 core-shell microspheres with mesoporous structures for highly efficient enrichment of phosphopeptides. We have demonstrated that the mesoporous ZrO2 layer dramatically improves the selective enrichment of phosphopeptides. Our approach allows for in-situ elution and sensitive identification of both mono-phosphorylated and multi-phosphorylated peptides in MALDI-TOF mass spectrometry, with the detection limit of down to the femtomole range. The target phosphopeptides can reliably be enriched for MS analysis from various complex samples including the spiked protein digests and tumor cell lysates. The Fe3O4@mZrO2 core-shell microspheres promise a useful tool for phosphoproteomics by allowing for highly efficient and selective enrichment of the crucial signaling regulators in a low abundance.

Abstract B28: MS-based HLA peptide discovery: Tumor neoantigens and biotherapeutic T-cell epitopes

Abstract To enable the molecular-level characterization of MHC- associated peptides for neoantigen discovery and identification of antigenic peptides on biotherapeutics, we have developed optimized workflows that allow the identification of MHC- presented epitopes. MHC Class I and II function to display peptide fragments of processed self and foreign proteins (antigens) on the cell surface for inspection by T cells (CD8+ cytotoxic T cells for Class I, and CD4+ helper T cells for Class II). Recognition of foreign antigens by T cell receptors triggers an immediate T-cell activation and expansion, resulting in the immune response. Characterizing these antigens is paramount to understanding the immunogenicity of proteins (e.g., biotherapeutic proteins) and generating tools for targeted cell destruction (e.g., cancer immunotherapy). Tumor neoantigens result from somatic mutations during oncogenesis and have immense immunotherapeutic value. Genomic sequencing of cancer tumor tissues combined with bioinformatics has enabled the identification of tumor specific mutations and in silico prediction of MHC-associated neoantigenic peptides. Often the results of the prediction algorithms are discordant with actual binding information. Neoantigen prediction by gene sequencing and in silico approaches can be complemented and further validated by empirical MHC peptide sequencing. Of interest to BioPharma is that biotherapeutics have the potential to exhibit immunogenicity, resulting in the expression of antidrug antibodies, which reduce drug load and effectiveness. Even though most protein therapeutics are humanized, single-nucleotide polymorphisms in endogenous related proteins can be the source of the immunogenic response. Thus, it is imperative to identify the biotherapeutic derived epitopes presented by MHC Class II to T helper cells with the goal of modifying the drug to remove the antigenic peptides without reducing efficacy. It also can lead to a personalized approach with multiple versions of the same drug mapped to the appropriate patient haplotype to insure efficacy without immunogenic response. The molecular-level characterization of peptides associated with MHC Class I and II requires MHC enrichment by immunoprecipitation, an unbiased peptide elution, and analysis by mass spectrometry. Here we present case studies of our recent work applying workflows for the analysis of peptides associated with Class I and Class II MHC molecules. Using human cell lines (HCT116 and Colo205), we present optimized methods for cell treatment/growth and lysis conditions, MHC immunoaffinity purification, peptide enrichment, mass spectrometry and data processing for sensitive and specific analysis of MHC-associated peptides. These assays have utility to improve prediction of neoantigen identification and mitigate risk of biotherapeutic immunogenicity. Citation Format: Michael Pisano, Paul Del Rizzo, James Mobley, Kamal Houssain, Bill Ho, Richard Jones, David Allen, Ravi Amunugama, Michael Ford. MS-based HLA peptide discovery: Tumor neoantigens and biotherapeutic T-cell epitopes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B28.