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Adaptation of an immunoaffinity chromatography protocol for the isolation of the cell line immunopeptides

ABSTRACT: BACKGROUND. For developing targeted immunotherapy against ovarian adenocarcinoma, direct identification of peptides presented by major histocompatibility complex class I (HLA I) on the surface of tumor cells can be utilized. However, standard immunopeptidomics protocols may vary substantially in their efficiency of immunopeptidome isolation depending on lysis conditions. Optimizing detergents for immunoaffinity purification of complexes enhances analytical sensitivity, expands the repertoire of identified ligands, and increases the likelihood of detecting clinically relevant peptides suitable for personalized immunotherapeutic approaches.AIM. To evaluate the effect of various detergents on the efficiency of identification of peptide ligands of HLA I complexes. METHODS. Immunopeptidome from cell lines were isolated using immunoaffinity chromatography and analyzed by liquid chromatography-mass spectrometry.RESULTS. In this study, we tested an affinity chromatography-based immunopeptidome isolation protocol, comparing various detergents (CHAPS, NP-40, SOD, and Triton X-100) for cell lysis, and observed no statistically significant differences in the number of identified peptides.CONCLUSION. Each of the four tested detergents provides identification of unique sets of peptides.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Georgij Arapidi

LAB HEAD: Georgij Arapidi

PROVIDER: PXD077080 | Pride | 2026-04-15

REPOSITORIES: Pride

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			Action	DRS
	P1032.dat	Other
	P1032.mgf	Mgf
	P1032.mzid	Mzid
	P1032.mzid_P1032.MGF	Mzid
	P1032.raw	Raw

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Adaptation of immunoaffinity protocol for the isolation of the cell line immunopeptides - immunopeptidomics part

Project description:BACKGROUND: The peptide repertoire presented by Human Leukocyte Antigens (HLA) provides a snapshot of intracellular proteins, which is monitored by the immune system. Although mass spectrometry is the gold standard for identification of these peptides, the upstream steps of the immunopeptidomics workflow, including immunoprecipitation and peptides purification, vary and critically impact the peptide yield.AIM: To optimize conditions for the isolation of a larger number of HLA I ligands.METHODS: Immunopeptidomes from cell lines were isolated using immunoaffinity followed by liquid chromatography-mass spectrometry analysis. Cell lines were lysed with various detergents (CHAPS, NP-40, SOD, Triton X-100) and eluted with TFA, urea, or thiocarbamate solutions.RESULTS: We optimized a protocol for immunoprecipitating peptide-HLA complexes and found that the number of identified peptides scales with the number of input cells, while being unaffected by the choice of lysis buffer.CONCLUSION: The optimized protocol allows the isolation of HLA I ligand peptides from suspension cell lines.

2026-04-15 | PXD077041 | Pride

Adaptation of immunoaffinity protocol for the isolation of the cell line immunopeptides - proteomics part

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Identification of major histocompatibility complex class I ligands in ovarian adenocarcinoma tumors

Project description:BACKGROUND. For developing targeted immunotherapy against ovarian adenocarcinoma, direct identification of peptides presented by major histocompatibility complex class I (HLA I) on the surface of tumor cells can be utilized.AIM. To identify ligands of HLA I of ovarian adenocarcinoma tumors. METHODS. Immunopeptidome from postoperative material of patients with ovarian adenocarcinoma were isolated using immunoaffinity chromatography and analyzed by liquid chromatography-mass spectrometry.RESULTS. Using NP-40, 5 peptides belonging to the proteins of cancer/testis antigens (CTA) were identified in the immunopeptidomes of postoperative material from patients with ovarian adenocarcinoma, 3 of which, according to the human protein atlas, are indeed not expressed in normal ovarian tissues.CONCLUSION. Immunopeptidome analysis allows the identification of peptides of CTA proteins, but a larger sample of postoperative material from patients with ovarian adenocarcinoma and experimental testing of the immunogenicity of the identified peptides are needed for further research.

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Lapatinib-Induced Enhancement Of Mitochondrial Respiration In HER2-Positive SK-BR-3 Cells: Mechanism Revealed By Analysis Of Proteomic But Not Transcriptomic Data

Project description:Dual inhibitors of HER2 and EGFR, such as lapatinib, have shown significant efficacy for the therapy of HER2-positive breast cancer. Previous experiments showed that in cell cultures, the efficacy of lapatinib was significantly reduced by exposure to human serum and human epidermal growth factor (EGF). At the proteomic and transcriptomic levels, we examined the changes in the HER2-positive breast cancer cell line SK-BR-3 profiles upon treatment with lapatinib, either alone or in combination with human serum or EGF. Proteomic profiling revealed 350 differentially expressed proteins (DEPs) in response to lapatinib treatment at concentrations that induced cell growth arrest. Addition of human serum or EGF in combination with lapatinib prevented cell growth inhibition, and this combination treatment returned the expression of ~93% of DEPs to drug-free levels for both human serum and EGF. Gene ontology enrichment and OncoboxPD pathway activation level analysis showed that lapatinib addition influenced mostly common functional processes revealed in RNA- and protein-based assays. However, a specific feature was observed at the proteome level: addition of lapatinib increased the expression of proteins associated with mitochondrial function and cellular respiration. This feature was not observed when using RNA sequencing data for the same experiments. However, it is consistent with the results of the resazurin test, which showed a 1.8-fold increase in SK-BR-3 cellular respiration upon exposure to lapatinib. Thus, we conclude that enhanced cellular respiration is a novel additional mechanism of action of lapatinib on HER2-positive cancer cells.

2024-09-26 | PXD054180 | Pride

Transcriptomics of HL60 cell line at 30 min and 1 h after all-trans-retinoic acid (ATRA) treatment

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Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles.

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