Dataset Information

The effect of proteasome inhibition on the HLA peptidome

ABSTRACT: The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Since the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and non-proteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides. Bioinformatics & data processing: Peptides were identified and the dynamic-SILAC data were quantified using the MaxQuant and the Proteome Discoverer software tools. Graphical representation of the bioinformatics results was performed with Perseus, version 1.3.0.4. MaxQuant [54] version 1.3.0.5 was used with the Andromeda search engine [55] and the human section of Nov 2011 of UniProt containing 20257 entries and mass tolerance of 6 ppm for the precursor masses and 0.5 Da for the fragments. Methionine oxidation was accepted as variable modification for both tryptic and HLA peptides. Carbamidomethyl cysteine and n-acetylation were accepted as modification for the proteomics analyses. Minimal peptide length was set to 7 amino acids and a maximum of two missed cleavages was allowed for tryptic peptides. The false discovery rate (FDR) was set for tryptic peptides to 0.01 for protein identifications, and 0.05 for the MHC peptides. The resulting identified protein tables were filtered to eliminate the identifications derived from the reverse database, as well as common contaminants. Proteome Discoverer version 1.3 (Thermo-Fisher) was used with UniProt of April 2012 for the Sequest search (containing 20220 entries) and July 2012 for the Mascot search (containing 20306 entries). Masses tolerance was set to 6 ppm for the precursors and 0.5 Da for the fragments. Methionine oxidation and n-acetylation were accepted as variable modification for both tryptic and HLA peptides. Carbamidomethyl cysteine was set as fixed modification for the proteomics analyses. Mass range of 350-5000 Da was used for tryptic peptides and mass range of 750-2500 Da was used for the MHC peptides. For both tryptic and MHC peptides the PSMs were filtered with at least 0.05 FDR (medium confidence), peptide maximum rank was set to 1. Minimal number of identified peptides per proteins was set to 2.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Arie Admon

LAB HEAD: Arie Admon

PROVIDER: PXD000171 | Pride | 2018-11-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Listofruns-MilnerGutterAdmonforMCP.xlsx	Xlsx
	Maxquant-AlltextfilesfromHLA-peptides.zip	Other
	Maxquant-AlltextfilesfromProteomics-longruns.zip	Other
	Maxuant-AlltextfilesfromProteomics-SCX.zip	Other
	Seq27808_E2O2.RAW	Raw

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Publications

The effect of proteasome inhibition on the generation of the human leukocyte antigen (HLA) peptidome.

Milner Elena E Gutter-Kapon Lilach L Bassani-Strenberg Michal M Barnea Eilon E Beer Ilan I Admon Arie A

Molecular & cellular proteomics : MCP 20130328 7

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we f ...[more]

PMID: 23538226

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