Carr - HLA_Melanoma_TSQQuantiva

Optimized liquid and gas phase fractionations increase HLA-peptidome coverage for primary cell and tissue samples
Data License: CC BY 4.0 | ProteomeXchange: PXD027165 | doi: https://doi.org/10.6069/yhnb-r803
  • Organism: Homo sapiens
  • Instrument: TSQ Quantis
  • SpikeIn: Yes
  • Keywords: HLA, targeted MS
  • Lab head: Steve Carr Submitter: Hasmik Keshishian
Abstract
Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require billions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limiting amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 50 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens
Experiment Description
HLA-I peptide enrichment and LC- MS/MS analysis Soluble lysates from up to 50 million single HLA-expressing B721.221 cells and up to 0.2 g from tumor samples were immunoprecipitated with W6/32 antibody (sc-32235, Santa Cruz) as described previously(Sarkizova et al. 2020). Iodoacetamide (10 mM) was added to the lysis buffer to alkylate cysteines. HLA-I bound peptides were acid eluted on 1cc 50mg tC18 SepPak Cartridges (WAT054960, Waters) on a vacuum manifold. Sep Paks are equilibrated with 200μL MeOH x 2, 100μL 50% ACN/1% FA, 500μL 1% FA x 4, respectively. Beads with HLA-I bound peptides were resuspended in 3% ACN/ 5% FA and transferred to the equilibrated cartridge. Peptides were eluted from HLA-I proteins with 10% acetic acid or 5 minutes twice and then desalted with 4x 500μL 1% FA. Finally, peptides were eluted from the desalt matrix using 250 μL 15% ACN/1% FA and 250μL 50% ACN/ 1% FA and dried down. Multiple reaction monitoring (MRM) for neoantigen detection Tier 2 multiple reaction monitoring (MRM) assay configuration using heavy labeled synthetic peptides was done on TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher) coupled with Easy-nLC 1200 ultra-high pressure liquid chromatography (UPLC) system (Thermo Fisher). Skyline Targeted Mass Spec Environment ((64-bit) 20.2.0.343 version)) was used throughout assay configuration and all data analysis. First, spectral library for the peptide YIHGRGWAL was generated on a Fusion Lumos (Thermo Fisher). Spectral library was uploaded to Skyline and 6 most intense fragment ions (transitions) were selected for MRM assay configuration. Next, collision energies (CE) were optimized for all the transitions a by liquid chromatography – multiple reaction monitoring mass spectrometry (LC-MRM/MS) on TSQ Quantiva using Skyline’s CE optimization module. For every transition starting with the instrument specific calculated CE we tested 10 additional CEs (5 below and 5 above the calculated CE) in increments of 2. The list of transitions with varying CE values was exported from Skyline and used for building MRM method in Xcalibur software. Equimolar mixture of peptides at 50fm/ul was analyzed by LC-MRM/MS on Quantiva using this method. Resulting data was analyzed on Skyline which then selected the CE that resulted in the highest peak area for each transition. In the final step of CE optimization MRM data was acquired with optimized CE values for every transition. Using this dataset in Skyline, the best 2 transitions were selected manually.After CE optimization, we analyzed three replicates of 6 IPs pooled with either 20 fmol or 40 fmol heavy peptide spiked in. Liquid chromatography was performed on a 75um ID picofrit columns packed in-house to a length of 28-30cm with Reprosil C18-AQ 1.9um beads (Dr Maisch GmbH) with solvent A of 0.1% formic acid (FA) / 3% acetonitrile (ACN) and solvent B of 0.1% FA / 90% ACN at 200nL/min flow rate and the same gradient as described above. MS parameters include 1.5 sec cycle time, Q1 and Q3 resolution of 0.4 and 0.7, respectively, RT scheduling window of 10min.
Sample Description
Tumor derived melanoma cell lines were lysed, HLA–peptide complexes were immunoprecipitated from 0.1–0.2 g tissue or up to 50 million cells. Solid tumor samples were dissociated using a tissue homogenizer (Fisher Scientific 150) and HLA complexes were enriched as described below.
Created on 7/9/21, 7:13 AM
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HLA_NeoAntg_HL_Mel6_PeptideYIH_2021-07-08_17-37-20.sky.zip2021-07-09 07:13:47112451