Summary

Title

Digital mapping of immunopeptidomes

Description

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.

Sample Processing Protocol

HLA class I molecules were isolated using standard immunoaffinity purification as described previously. In brief, snap-frozen cell pellets were lysed in 10 mM CHAPS/PBS (AppliChem, St Louis, MO, USA/Gibco, Carlsbad, CA, USA) containing 1x protease inhibitor (Complete; Roche, Basel, Switzerland). HLA molecules were single-step purified using the pan-HLA class I-specific mAb W6/32, which was covalently linked to CNBr-activated sepharose (GE Healthcare, Chalfont St Giles, UK). HLA–peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (Merck, Whitehouse Station, NJ, USA). Elution fractions E1–E8 were pooled and free HLA peptides were isolated by ultrafiltration using centrifugal filter units (Amicon; Millipore, Billerica, MA, USA). HLA peptides were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Millipore). Extracted peptides were eluted in 35 μl of 80% acetonitrile (Merck)/0.2% trifluoroacetic acid, centrifuged to complete dryness and resuspended in 25 μl of 1% acetonitrile/0.05% trifluoroacetic acid. Samples were stored at − 20 °C until analysis by LC-MS/MS. Three different MS were used for the generation of the spectral libraries: an AB SCIEX TripleTOF 5600+, a Thermo Scientific Orbitrap XL and a Thermo Scientific Orbitrap ELITE.

Data Processing Protocol

The datasets were searched individually using X!tandem, MS-GF+ and Comet against the full non-redundant, canonical human genome as annotated by the UniProtKB/Swiss-Prot (2014_02) with 20,270 ORFs and appended iRT peptide and decoy sequence. Oxidation (M) was the only variable modification. Parent mass error was set to ±5p.p.m., fragment mass error was set to ±0.5Da. The search identifications were then combined and statistically scored using PeptideProphet and iProphet within the TPP.

Contact

Etienne Caron, ETH Zurich
Ruedi Aebersold, ETH Zurich Institute of Molecular Systems Biology ( lab head )

Submission Date

06/03/2015

Publication Date

14/07/2015

Cell Type

B cell
T lymphocyte

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    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. An open-source computational and data resource to analyze digital maps of immunopeptidomes. Elife. 2015 Jul 8;4 PubMed: 26154972