Dataset Information

Global Identification of Post-Translationally Spliced Peptides with Neo-Fusion.

ABSTRACT: Post-translationally spliced peptides have recently garnered significant interest as potential targets for cancer immunotherapy and as contributors to autoimmune diseases such as type 1 diabetes, yet feasible identification methods for spliced peptides have yet to be developed. Here we present Neo-Fusion, a search program for discovering spliced peptides in tandem mass spectrometry data. Neo-Fusion utilizes two separated ion database searches to identify the two halves of each spliced peptide, and then it infers the full spliced sequence. This strategy allows for the identification of spliced peptides without peptide length constraints, providing a broadly applicable tool suitable for identification of spliced peptides in a variety of systems, such as the HLA-I and HLA-II immunopeptidomes and in vitro digested protein samples obtained from organelles, cells, or tissues of interest. Using simulated spliced peptides to benchmark Neo-Fusion, 25% of all simulated spliced peptides were identified at a measured false-discovery rate of 5% for HLA-I. Neo-Fusion provides the research community with a powerful new tool to aid in the study of the prevalence and biological significance of post-translationally spliced peptides.

SUBMITTER: Rolfs Z

PROVIDER: S-EPMC6465104 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Global Identification of Post-Translationally Spliced Peptides with Neo-Fusion.

Rolfs Zach Z Solntsev Stefan K SK Shortreed Michael R MR Frey Brian L BL Smith Lloyd M LM

Journal of proteome research 20181031 1

Post-translationally spliced peptides have recently garnered significant interest as potential targets for cancer immunotherapy and as contributors to autoimmune diseases such as type 1 diabetes, yet feasible identification methods for spliced peptides have yet to be developed. Here we present Neo-Fusion, a search program for discovering spliced peptides in tandem mass spectrometry data. Neo-Fusion utilizes two separated ion database searches to identify the two halves of each spliced peptide, a ...[more]

PMID: 30346791

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Project description:Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8-12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5-7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-021-00066-x.

Dataset Information

Global Identification of Post-Translationally Spliced Peptides with Neo-Fusion.

Publications

Global Identification of Post-Translationally Spliced Peptides with Neo-Fusion.

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