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Tissue expression of N-terminal proteoforms.

ABSTRACT: To evaluate the expression of N-terminal proteoforms (protein isoforms with different N-terminus expressed from the same gene) in healthy human tissues, we re-analyzed public proteomics data of the draft map of the human proteome developed by the Pandey group (PRIDE project PXD000561). Using ionbot search engine and a custom-build protein sequence database (composed of UniProt and Ribo-seq derived proteoforms) we confirmed that N-terminal proteoforms are not only expressed in histologically healthy human tissues, but also display tissue specificity.

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart, Testis, Brain, Prostate Gland, Gastrointestinal Tract, Liver, Lung, Blood, Kidney, Ovary, Placenta, Nerve Cord, Colon, Urinary Bladder, Adrenal Gland

SUBMITTER: Daria Fijalkowska

LAB HEAD: Prof. Dr. Kris Gevaert

PROVIDER: PXD039339 | Pride | 2023-06-12

REPOSITORIES: Pride

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			Action	DRS
	Adult_Adrenalgland_Gel_Elite_49_f01.raw	Raw
	Adult_Adrenalgland_Gel_Elite_49_f02.raw	Raw
	Adult_Adrenalgland_Gel_Elite_49_f03.raw	Raw
	Adult_Adrenalgland_Gel_Elite_49_f04.raw	Raw
	Adult_Adrenalgland_Gel_Elite_49_f05.raw	Raw

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Project description:Infection or reactivation with human cytomegalovirus (HCMV) is still a major cause of morbidity and mortality in patients undergoing solid organ transplantation or allogeneic stem cell transplantation (alloSCT). Recent studies have shown that protection against latent HCMV infection relies on a much broader antigen spectrum than previously expected. The identification of novel immunogenic HCMV-derived peptides that facilitate HCMV control is therefore essential to improve immune monitoring of HCMV-specific T cells and HCMV vaccine development. In particular, only a few HCMV-derived peptides have been described for less common HLA alleles, such as HLA-A*03:01 and HLA-B*15:01, limiting the implementation of these techniques for a substantial number of patients. Here, we identified novel HCMV-derived HLA-A*03:01- and HLA-B*15:01-restricted immunogenic peptides by an innovative combined in vitro and in silico approach. We utilized the computational tools Peptide-PRISM (1) and PRICE (2) to analyze mass spectrometric (MS) and ribosome sequencing (ribo-seq) datasets, respectively. To refine our in silico approach, we utilized machine learning to rate the identified peptide candidates based on their translational activity, binding affinity and positioning within the small open reading frames (sORFs), with the goal of assessing their likelihood to be presented on HLA-A*03:01 and HLA-B*15:01 molecules. After identifying the highest-scoring 49 canonical and 49 cryptic potentially immunogenic peptides for each HLA allele, we assessed their immunogenicity by screening HCMV-seropositive and -seronegative healthy donors, as well as alloSCT patients for their peptide-specific T-cell responses. We used a stimulation procedure in two consecutive steps: peripheral blood cells (PBMCs) were stimulated starting with peptide pools and subsequently with potentially reactive single peptides. In vitro T-cell stimulation resulted in the direct identification of three canonical and one cryptic HLA-A*03-restricted immunogenic peptides as well as five canonical and one cryptic HLA-B*15-restricted immunogenic peptide, with a specific IFNγ+/CD8+ T cell response of ≥ 0.02%. Highest T-cell responses were identified against two HLA-A*03-restricted and three HLA-B*15-restricted canonical peptides with up to 3.42% of IFNγ+/CD8+ T cells in patients 180 days post alloSCT. In conclusion, we identified specific T-cell responses against eight canonical and two cryptic HLA-A*03:01- and HLA-B*15:01-restricted peptides in healthy individuals and post-transplanted patients and which were classified as immunogenic. Of these, six peptides are novel HCMV-derived, immunogenic peptides according to the UniProt database, broadening the spectrum of immunogenic HCMV-derived peptides for personalized immune monitoring and vaccine development.

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Tissue expression of N-terminal proteoforms.

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