Why Immunopeptidome Profiling?

The immunopeptidome—comprising peptides naturally presented by HLA molecules—serves as a real-time molecular signature of cellular state. It reveals changes driven by mutations, infections, or immune dysregulation long before phenotypic symptoms appear.

By profiling these HLA-bound peptides, researchers gain an actionable readout of the immune system’s interaction with tumor cells, pathogens, or self-antigens.

What Makes Immunopeptidomics Powerful?

Unlike transcriptomics or proteomics, immunopeptidomics directly captures what the immune system can "see"—the actual surface-displayed peptides recognized by T cells. It bridges the gap between expression and immune recognition.

Key Application Areas

When paired with genomics, transcriptomics, and proteomics, immunopeptidomics adds a functional layer that reveals how molecular changes translate into immune-detectable differences—crucial for drug discovery, biomarker validation, and precision immunotherapy.

Cancer Immunotherapy

Identify tumor-specific and mutated peptides (neoantigens) for designing personalized vaccines, TCR therapies, and immune checkpoint strategies.

Infectious Disease

Reveal pathogen-derived peptides actively presented on infected cells to inform epitope-based vaccine development and antiviral drug targeting.

Autoimmune Research

Characterize self-peptides implicated in T cell misrecognition, enabling the study of molecular mimicry and autoimmune triggers.

What We Offer: Comprehensive Immunopeptidome Profiling and Beyond

Creative Proteomics provides an end-to-end immunopeptidomics service for the in-depth characterization of naturally presented HLA-bound peptides. By integrating advanced mass spectrometry platforms, custom enrichment workflows, and sophisticated bioinformatics pipelines, we empower researchers to identify, quantify, and interpret the dynamic antigen landscape in both physiological and disease states.

What We Can Analyze

HLA Class I Peptidome

Comprehensive coverage of intracellular and nuclear-derived peptides (8–12mers), ideal for CD8⁺ T cell epitope studies.

HLA Class II Peptidome

Profiling of extracellular, endocytic, and autophagy-derived peptides (13–25mers) relevant to CD4⁺ T cell immunity.

Post-Translationally Modified Peptides

Identification of phosphorylated, glycosylated, citrullinated, and other modified epitopes without prior enrichment.

Non-Canonical / Cryptic Peptides

Detection of peptides derived from lncRNAs, introns, upstream ORFs, repeat elements, or alternative splicing.

Treatment-Responsive Peptidome

Assess the immunopeptidomic changes under cytokine stimulation, drug exposure, gene editing, or environmental perturbations.

Neoantigen Candidate Discovery

Extraction and prioritization of mutation-derived HLA ligands for vaccine design or T cell immunotherapy development.

Specialized Add-On Modules (Optional)

Immunopeptide Affinity Screening

High-throughput screening of peptides against target antigens using ELISA, surface plasmon resonance (SPR), or molecular docking. Ranking based on affinity and specificity.

Custom Immune Peptide Library Construction

Design and synthesis of diverse peptide libraries optimized for immunogenicity, PTM coverage, or structure-based selection.

Multiplexed MS Quantification (TMT/iTRAQ)

Isobaric labeling for comparative peptidomics across treatment arms or patient cohorts.

HLA Binding Motif Prediction & Prioritization

Includes motif clustering, allele assignment, and anchor residue analysis.

Platform Advantages

Low Sample Input Compatibility

Requires as little as 1×10⁶ cells or ~5 mg of tissue; suitable for frozen, fresh, or limited biopsy samples.

Dual-Class HLA Analysis

Simultaneous profiling of HLA-I and HLA-II peptides from the same sample.

HLA Binding Motif Characterization

Motif deconvolution and editing pattern analysis for tapasin-, ERAP1/2-, HLA-DM/DO-dependent regulation.

Quantification-Ready Workflows

Supports DDA, DIA, and PRM/SureQuant for both relative and absolute quantification.

HLA Deconvolution & Allele Assignment

Compatible with monoallelic, multiallelic, and unknown donor samples; supports allele-specific peptide clustering.

Comprehensive Bioinformatics Outputs

Includes peptide ID lists, motif logos, binding affinities, source protein mapping, and pathway enrichment (upon request).

Step-by-Step Immunopeptidome Profiling Workflow

Deep and Accurate Peptidome Discovery

At Creative Proteomics, our immunopeptidomics workflow is powered by next-generation Orbitrap mass spectrometry platforms—Orbitrap Astral™ and Orbitrap Exploris™ 480—ensuring exceptional sensitivity, resolution, and throughput for comprehensive HLA ligand profiling.

Whether you're working with rare cell types or large cohorts, our platform delivers reproducible, deep-coverage datasets that accelerate epitope discovery and immunological insight.

Technical Highlights

• >90% MS/MS Coverage
  High-resolution tandem MS enables identification of thousands of HLA-bound peptides with superior confidence and spectral clarity.
• 1% FDR Stringent Filtering
  Strict false discovery rate (FDR) control ensures reliable peptide identification and reproducibility across biological replicates.
• PRM & SureQuant™-Enabled Quantification
  Supports both relative and absolute quantification, with enhanced sensitivity for low-abundance immune peptides.
• Broad Input Range: 1M–100M Cells
  Optimized for scarce biospecimens or bulk materials—our platform scales with your project’s needs.

Sample Requirements for Immunopeptidomes

Demo Results

Deliverables | What You Will Receive

• Identified Peptides (CSV Format)
  List of all HLA-bound peptides with sequences, lengths, charge states, and spectral quality scores.
• Quantitative Data (Normalized)
  Relative peptide abundance (intensity or spectral count) across sample groups, ready for statistical analysis or visualization.
• HLA Binding Motif Reports
  Sequence logos and binding motif tables derived from deconvoluted peptide sets, categorized by HLA alleles.
• PTM and Variant Annotations (if applicable)
  Detected phosphorylation, glycosylation, or citrullination sites, and candidate neoantigen peptides with mutation context.
• Pathway & Ontology Enrichment (Optional)
  Functional insights into peptide source proteins, including GO terms, KEGG pathways, and immune-related processes.
• Graphical Summary PDF
  A visual executive summary with peptide length distribution, volcano plots, and key motif logos.

FAQ for Immunopeptidome Analysis

Summary

Using advanced mass spectrometry and a novel spliced peptide prediction pipeline, researchers analyzed immunopeptidomic data from cancer patients and cell lines. They identified over 150 recurrent posttranslational spliced peptides presented by HLA class-I and class-II molecules, several of which derived from cancer-related genes like DAPK1 and HLA-E. Importantly, two of these peptides were validated using synthetic standards and shown to elicit CD8⁺ T cell immune responses, highlighting their potential as immunotherapy targets.

Methods

This study presents a novel computational pipeline combining Neo-Fusion and MaxQuant to stringently identify and validate recurrent PTSPs across multiple cancer types using large-scale MS-based immunopeptidomics data.

Key Technical Features:

• Sample types: Patient-derived melanoma lines, breast cancer (MCF7), HNSCC (SCC47), and mouse melanoma clones
• Peptide identification: Neo-Fusion detects spliced sequences; MaxQuant performs spectrum matching
• Stringent filtration: Peptides were filtered for binding affinity (NetMHCpan), MS/MS coverage, retention time, transcriptomic origin (RNA-seq), and presence in normal or microbial proteomes
• Validation: Light and heavy synthetic peptides were used to confirm MS identifications
• Immunogenicity tests: Conducted using TILs and PBMC-derived T cells
• Recurrent peptides: Defined by presence across multiple independent patients or metastases

Creative Proteomics can offer advanced immunopeptidomics services that mirror and support this research pipeline, including:

• HLA peptidomics profiling using high-resolution LC-MS/MS
• Neoantigen discovery and PTSP identification
• HLA class-I and class-II ligandome mapping
• Quantitative and comparative immunopeptidome analysis across samples or treatment conditions
• Synthetic peptide validation and custom bioinformatics pipelines for spliced peptide filtering

These services empower cancer immunotherapy research, vaccine target screening, and immune evasion mechanism studies.

Results

PTSP Frequency Is Low but Significant

• PTSPs constitute <3.1% of HLA class-I and <0.5% of HLA class-II immunopeptidomes.
• Despite the low proportion, PTSPs outnumber neoantigens derived from somatic mutations, highlighting their potential clinical relevance.

Recurrent PTSPs Identified Across Patients

• 143 HLA class-I and 10 HLA class-II recurrent PTSPs were discovered.
• These peptides are associated with cancer-related genes such as MITF, DAPK1, IL17RE, and HLA-E.

Immunogenicity Validated

• Two PTSPs (from DAPK1 and HLA-E) elicited significant CD8+ T cell responses in autologous TILs and PBMC-derived T cells.
• Flow cytometry showed upregulation of activation markers (e.g., 4-1BB, IFN-γ, TNF-α).
• Dextramer staining confirmed peptide-specific CD8+ T cell populations in healthy donors.

Spliced Peptides Recur in Single-Cell Clones

• Spliced peptides were consistently detected across 5–15 single-cell clones derived from the same mouse tumor, demonstrating biological reproducibility.

Pipeline Validation and Stringency

• Most spurious hits were removed by Delta score and Leu/Ile ambiguity filters.
• Leu/Ile replacement filter alone provided a 30–40× discrimination between WT and spliced peptide false-positives.

Overlay of endogenous and isotopically labeled HLA-E–derived peptide (WSDSSGGKGGSY) chromatograms in targeted immunopeptidomics of 83T melanoma cells, visualized via Skyline.

Combined Omics Approaches Reveal Resistance Mechanisms to Beet Curly Top Virus in Sugar Beet Genotypes

BRCA1 Antibodies Matter: Benchmarking Reagent Specificity in Cancer Research

In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes

Trypanosoma cruzi DNA Polymerase β Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2)

The Interplay Between GSK3β and Tau Ser262 Phosphorylation During the Progression of Tau Pathology

Inactivation of Myeloperoxidase by Benzoic Acid Hydrazide