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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Georgij Arapidi

LAB HEAD: Georgij Arapidi

PROVIDER: PXD076866 | Pride | 2026-04-15

REPOSITORIES: Pride

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			Action	DRS
	070521P270.dat	Other
	070521P270.mgf	Mgf
	070521P270.mzid	Mzid
	070521P270.mzid_070521P270.MGF	Mzid
	070521P270.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.

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

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. 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.

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