Dataset Information

MS-rescue: A Computational Pipeline to Increase the Quality and Yield of Immunopeptidomics Experiments

ABSTRACT: We propose a new pipeline for the refinement of spectrum-peptide assignment, designed specifically for MHC ligand identification. By modeling the peptidome as a collection of a limited number of specificities, corresponding to the MHC alleles of the cell line, our method achieves increased sequencing depth, while at the same time removing potential experimental outliers and contaminants.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

SUBMITTER: Nicola Ternette

LAB HEAD: Nicola Ternette

PROVIDER: PXD011723 | Pride | 2019-01-02

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	FL605_IPP75_AN_Jurkat_C076_1BR_Even.mgf	Mgf
	FL605_IPP75_AN_Jurkat_C076_1BR_Even.raw	Raw
	FL605_IPP75_AN_Jurkat_C076_1BR_Odd.mgf	Mgf
	FL605_IPP75_AN_Jurkat_C076_1BR_Odd.raw	Raw
	FL605b_IPP75_AN_Jurkat_C076_1BR_Even.mgf	Mgf

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Publications

MS-Rescue: A Computational Pipeline to Increase the Quality and Yield of Immunopeptidomics Experiments.

Andreatta Massimo M Nicastri Annalisa A Peng Xu X Hancock Gemma G Dorrell Lucy L Ternette Nicola N Nielsen Morten M

Proteomics 20190118 4

LC-MS/MS has become the standard platform for the characterization of immunopeptidomes, the collection of peptides naturally presented by major histocompatibility complex molecules to the cell surface. The protocols and algorithms used for immunopeptidomics data analysis are based on tools developed for traditional bottom-up proteomics that address the identification of peptides generated by tryptic digestion. Such algorithms are generally not tailored to the specific requirements of MHC ligand ...[more]

PMID: 30578603

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Project description:Human leukocyte antigen (HLA) - B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are two strong predisposing genetic factors to Behçet's disease (BD). Previous studies have focused on two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry. The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2. This unconventional group of HLA-B*51:01-bound peptides was enriched for 8-mers (with fewer 9-mers) compared to Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides with the exception of the less abundant Leucine at position Ω. Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type - dependent manner. The HLA-B*51:01 peptidome includes a surprisingly high proportion of unconventional non-Pro/Ala2 peptides which are increased by ERAP1 silencing, mimicking the loss-of-function BD risk variant.