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An open-source computational and data resource to analyze digital maps of immunopeptidomes.

Caron E, Espona L, Kowalewski DJ, Schuster H, Ternette N, Alpízar A, Schittenhelm RB, Ramarathinam SH, Lindestam Arlehamn CS, Chiek Koh C, Gillet LC, Rabsteyn A, Navarro P, Kim S, Lam H, Sturm T, Marcilla M, Sette A, Campbell DS, Deutsch EW, Moritz RL, Purcell AW, Rammensee HG, Stevanovic S, Aebersold R - Elife (2015)

Bottom Line: We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides.Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS).This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zurich, Switzerland.

ABSTRACT
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

No MeSH data available.


Reproducibility of DDA and SWATH-MS for the identification of HLA class I peptides.HLA class I peptides were isolated from JYEBV+ cells. Six technical replicates were consecutively injected in a TripleTOF 5600 MS. The Venn diagrams indicate the number of peptides identified in each analysis and the number of peptides shared between the runs. (A) Three datasets were acquired in DDA mode and the peptides were identified using the open source database search engines (1% peptide-level FDR). (B) Three datasets were acquired in SWATH mode and the peptides were identified using OpenSWATH and a combined HLA-A and -B peptide assay library (1% peptide-level FDR).DOI:http://dx.doi.org/10.7554/eLife.07661.005
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fig1s1: Reproducibility of DDA and SWATH-MS for the identification of HLA class I peptides.HLA class I peptides were isolated from JYEBV+ cells. Six technical replicates were consecutively injected in a TripleTOF 5600 MS. The Venn diagrams indicate the number of peptides identified in each analysis and the number of peptides shared between the runs. (A) Three datasets were acquired in DDA mode and the peptides were identified using the open source database search engines (1% peptide-level FDR). (B) Three datasets were acquired in SWATH mode and the peptides were identified using OpenSWATH and a combined HLA-A and -B peptide assay library (1% peptide-level FDR).DOI:http://dx.doi.org/10.7554/eLife.07661.005

Mentions: Mass spectrometry (MS) has evolved as the method of choice for the exploration of the human immunopeptidome (Hunt et al., 1992; Admon and Bassani-Sternberg, 2011; Granados et al., 2015). The largest HLA peptidomes reported to date using MS contain more than 10,000 class I or class II peptides (Hassan et al., 2013; Bergseng et al., 2014; Bassani-Sternberg et al., 2015). Estimates from various analytical and cell-based techniques also indicate that individual peptides are expressed on average at 50 copies per cell with extremes ranging from 1 to 10,000 copies per cell (Granados et al., 2015). Until recently, the most common strategy for the analysis of immunopeptidomes by MS has focused on the isolation of HLA-bound peptides by immunoaffinity chromatography and the collection of fragment ion spectra of selected peptides through automated MS operated in data-dependent acquisition (DDA) mode. Although DDA is a powerful strategy for exploring the peptidomic content of various cell and tissue types, it is not a reliable platform for solving problems that require the comparison of comprehensive, quantitative, and reproducible data sets across many samples or conditions. In fact, analyses of complex/unfractionated digests of cell lysate using DDA have shown that as many as 84% of peptides may remain unselected for fragmentation even though they are clearly detectable by the mass spectrometer (Michalski et al., 2011). Although the complexity of isolated HLA peptides is hardly comparable with that of cell lysate digests, as many as 20% of the selected HLA peptides can vary between replicate analyses of the same sample (Granados et al., 2014) (Figure 1—figure supplement 1A). A second strategy, referred to as selected/multiple reaction monitoring (S/MRM), is a targeting MS technique capable of generating highly reproducible, quantitatively accurate and sensitive datasets (Picotti and Aebersold, 2012). S/MRM is, however, limited by its capacity to detect only tens to hundreds of peptides per sample injection and thus is not ideally suited to comprehensively quantify HLA peptidomes. To overcome this limitation, we recently introduced SWATH-MS, a new mass spectrometric technique that combines data-independent acquisition (DIA) with a targeted data extraction strategy (Gillet et al., 2012; Röst et al., 2014). In DIA mode, all peptides in a sample are fragmented and the corresponding fragment ion spectra are acquired, resulting in a digital recording of the peptide sample. DIA is an unbiased MS technique and therefore represents a suitable strategy for efficiently generating consistent, reproducible and quantitatively accurate measurements of peptides across multiple samples (Gillet et al., 2012; Collins et al., 2013; Rosenberger et al., 2014; Röst et al., 2014; Guo et al., 2015; Liu et al., 2015; Schubert et al., 2015a).


An open-source computational and data resource to analyze digital maps of immunopeptidomes.

Caron E, Espona L, Kowalewski DJ, Schuster H, Ternette N, Alpízar A, Schittenhelm RB, Ramarathinam SH, Lindestam Arlehamn CS, Chiek Koh C, Gillet LC, Rabsteyn A, Navarro P, Kim S, Lam H, Sturm T, Marcilla M, Sette A, Campbell DS, Deutsch EW, Moritz RL, Purcell AW, Rammensee HG, Stevanovic S, Aebersold R - Elife (2015)

Reproducibility of DDA and SWATH-MS for the identification of HLA class I peptides.HLA class I peptides were isolated from JYEBV+ cells. Six technical replicates were consecutively injected in a TripleTOF 5600 MS. The Venn diagrams indicate the number of peptides identified in each analysis and the number of peptides shared between the runs. (A) Three datasets were acquired in DDA mode and the peptides were identified using the open source database search engines (1% peptide-level FDR). (B) Three datasets were acquired in SWATH mode and the peptides were identified using OpenSWATH and a combined HLA-A and -B peptide assay library (1% peptide-level FDR).DOI:http://dx.doi.org/10.7554/eLife.07661.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507788&req=5

fig1s1: Reproducibility of DDA and SWATH-MS for the identification of HLA class I peptides.HLA class I peptides were isolated from JYEBV+ cells. Six technical replicates were consecutively injected in a TripleTOF 5600 MS. The Venn diagrams indicate the number of peptides identified in each analysis and the number of peptides shared between the runs. (A) Three datasets were acquired in DDA mode and the peptides were identified using the open source database search engines (1% peptide-level FDR). (B) Three datasets were acquired in SWATH mode and the peptides were identified using OpenSWATH and a combined HLA-A and -B peptide assay library (1% peptide-level FDR).DOI:http://dx.doi.org/10.7554/eLife.07661.005
Mentions: Mass spectrometry (MS) has evolved as the method of choice for the exploration of the human immunopeptidome (Hunt et al., 1992; Admon and Bassani-Sternberg, 2011; Granados et al., 2015). The largest HLA peptidomes reported to date using MS contain more than 10,000 class I or class II peptides (Hassan et al., 2013; Bergseng et al., 2014; Bassani-Sternberg et al., 2015). Estimates from various analytical and cell-based techniques also indicate that individual peptides are expressed on average at 50 copies per cell with extremes ranging from 1 to 10,000 copies per cell (Granados et al., 2015). Until recently, the most common strategy for the analysis of immunopeptidomes by MS has focused on the isolation of HLA-bound peptides by immunoaffinity chromatography and the collection of fragment ion spectra of selected peptides through automated MS operated in data-dependent acquisition (DDA) mode. Although DDA is a powerful strategy for exploring the peptidomic content of various cell and tissue types, it is not a reliable platform for solving problems that require the comparison of comprehensive, quantitative, and reproducible data sets across many samples or conditions. In fact, analyses of complex/unfractionated digests of cell lysate using DDA have shown that as many as 84% of peptides may remain unselected for fragmentation even though they are clearly detectable by the mass spectrometer (Michalski et al., 2011). Although the complexity of isolated HLA peptides is hardly comparable with that of cell lysate digests, as many as 20% of the selected HLA peptides can vary between replicate analyses of the same sample (Granados et al., 2014) (Figure 1—figure supplement 1A). A second strategy, referred to as selected/multiple reaction monitoring (S/MRM), is a targeting MS technique capable of generating highly reproducible, quantitatively accurate and sensitive datasets (Picotti and Aebersold, 2012). S/MRM is, however, limited by its capacity to detect only tens to hundreds of peptides per sample injection and thus is not ideally suited to comprehensively quantify HLA peptidomes. To overcome this limitation, we recently introduced SWATH-MS, a new mass spectrometric technique that combines data-independent acquisition (DIA) with a targeted data extraction strategy (Gillet et al., 2012; Röst et al., 2014). In DIA mode, all peptides in a sample are fragmented and the corresponding fragment ion spectra are acquired, resulting in a digital recording of the peptide sample. DIA is an unbiased MS technique and therefore represents a suitable strategy for efficiently generating consistent, reproducible and quantitatively accurate measurements of peptides across multiple samples (Gillet et al., 2012; Collins et al., 2013; Rosenberger et al., 2014; Röst et al., 2014; Guo et al., 2015; Liu et al., 2015; Schubert et al., 2015a).

Bottom Line: We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides.Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS).This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zurich, Switzerland.

ABSTRACT
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

No MeSH data available.