Summary

Title

Sampling from proteome to HLA-DR ligandome

Description

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, due to the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly modest correlation between the abundance levels of surface-presented peptides and the cellular expression of their source proteins. Important selective sieving from the sampled proteome to the ligandome, was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease.

Sample Processing Protocol

The MUTZ-3 cell line, an HLA-DR10, -DR11, -DR52 (HLA-DRB1*10, HLA-DRB1*11, HLA-DRB3*01) positive acute myelo-monocytic leukemia, was grown under maintenance conditions in roller bottles in α-Minimum Essential Medium (Gibco), supplemented 20% heat-inactivated FBS (Hyclone), 100 U/ml penicillin, 100 ug/ml streptomycin (Gibco), 2 mM L-glutamine (Gibco), 50 µM 2-mercaptoethanol (Serva) and 25 U/ml GM-CSF (Prepotech)[23, 24]. MUTZ-3 cells were induced into an immature DC state by a 5-day exposure to 1000 U/ml GM-CSF (100 ng/ml), 1000 U/ml IL-4 (20 ng/ml) and 2.5 ng/ml TNF-α. Immature MUTZ-3 DC were matured by increasing the concentration of TNFa to 75 ng/ml for 20 hr. During this maturation phase BCG antigens were present as an antigenic pulse with. The large bulk of 1.2x 109 cells stimulated MUTZ-3 was washed in ice cold PBS and snap frozen before lysing and solubilizing of cell membrane proteins with Nonidet P40 containing IP lysisbuffer (Thermo Scientific). After removal of the non-solubilized fraction using ultracentrifugation, HLA class II molecules were immunoprecipitated from the MUTZ-3 cell lysate using the HLA-DR-specific monoclonal antibody B8.11.2, as described previously[12]. An aliquot of the MUTZ-3 cell lysate after HLA-DR pull down was used for proteomics. HLA class II molecules and associated peptides were eluted with 10% acetic acid and peptides were collected by passage over a 10-kDa mw cutoff membrane and concentrated using vacuum centrifugation. The MUTZ-3 cell lysate was diluted in 2 M urea, 50 mM ammonium bicarbonate containing one tablet of EDTA-free protease inhibitor mixture (Sigma) and one tablet of PhosSTOP phosphatase inhibitor mixture (Roche). Cysteine residues were reduced and alkylated using 200 mM dithiotreitol (Sigma) and 200 mM iodoacetamide (Sigma). The proteins were digested with Lys-C (Roche Diagnostics) at an enzyme :protein ratio of 1:75 for 4 h at 37 °C. Two times diluted samples were digested with trypsin (Roche Diagnostics) overnight at 37°C at an enzyme : protein ratio of 1:100. Peptide mixtures were desalted using a 1-cc Sep Pack C18 columns (Waters) according manufacture’s protocol. HLA-DR eluted peptides were fractionated by strong cation exchange (SCX) chromatography[27]. The peptides were separated by a linear salt gradient ramping to 500 mM KCl in 0.1M HOAc and 35% acetonitrile at a column flow rate of 2 μl/min. A total number of 26 fractions (2 min per fraction) were collected, dried down using a vacuum centrifuge and reconstituted. Tryptic peptides from the MUTZ-3 digest were fractioned by SCX using a ZorbaxBioSCX-Series II column (0.8 mm I.D., 50 mm, 3.5 µm particle size, Agilent Technologies). A multistep gradient up to 500 mM Nacl in 0.05% formic acid 20% acetonitrile was used to separate the tryptic peptides[28]. For the HLA ligands, each individual SCX fraction was analyzed in triplicate by nanoscale LC-MS using a Thermo Scientific EASY-nLC 1000 (Thermo Fisher Scientific, Odense, Denmark) and ETD enabled LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with either EThcD, HCD or ETD fragmentation[26]. The column effluent was directly electro-sprayed into the MS using a gold-coated fused silica tapered tip of ~5 m I.D. Full MS spectra (m/z 300 to 1,500) were acquired in the Orbitrap at 60,000 resolution (FWHM). The 10 most abundant precursor ions were selected for either data-dependent EThcD, HCD or ETD fragmentation (isolation width of 1.5 Th) at an abundance threshold of 500 counts. Fragment ions were detected in the Orbitrap analyzer at 15,000 resolution (FWHM). The automatic gain control (AGC) target in MS/MS was set to 3 × 105 for EThcD, 7 × 104 for HCD, and 1 × 105 ETD. The maximum ion accumulation time for MS scans was set to 250 ms and for MS/MS scans to 1500 ms. For EThcD, modified instrument firmware was used to allow all-ion HCD fragmentation after an initial ETD. The HCD normalized collision energy was set to 32%. The ETD reaction time was set to 50 ms and supplemental activation and charge dependent activation time was enabled. Precursor ions with unknown and +1 charge states were excluded from MS/MS analysis. Dynamic exclusion was enabled (exclusion size list 500) with a repeat count of 1 and an exclusion duration of 60 s. The SCX fractions of the tryptic digested MUTZ-3 cells were analyzed by LC-MS/MS using an Agilent 1290 Infinity System (Agilent Technologies, Waldbronn, DE) modified for nanoflow LC (passive split) connected to a TripleTOF analyzer (AB Sciex). A voltage of 2.7 kV was applied to the needle. The survey scan was from 375 to 1250 m/z and the high resolution mode was utilized, reaching a resolution of up to 40,000. Tandem mass spectra were acquired in high sensitivity mode with a resolution of 20,000. The 20 most intense precursors were selected for subsequent fragmentation using an information dependent acquisition, with a minimum acquisition time of 50 ms.

Data Processing Protocol

Data analysis The raw files collected from the TripleTOF were first recalibrated based on five background ions with m/z values of 391.2847, 445.12003, 51913882, 593.15761, 667.17640. The calibrated raw files were converted to mgf by the AB Sciex MS Data Converter (version 1.3 beta) program before analysis with Proteome Discoverer 1.4. RAW files acquired with the Orbitrap Elite were directly analyzed with Proteome Discoverer 1.4 software package (Thermo Fisher Scientific, Bremen, Germany) using default settings unless otherwise stated. For the EThcD and ETD spectra the non-fragment filter was added with the following settings: the precursor peak was removed within a 1 Da window, charged reduced precursors and neutral loss peaks were removed within a 0.5 Da window. MS/MS scans were searched against the human Uniprot database using the SEQUEST HT mode (Proteome Discoverer 1.4, Thermo Fisher Scientific). Precursor ion and MS/MS tolerances were set to 3 ppm and 0.02 Da, respectively. In SEQUEST, spectrum matching was set to one for c and z ions for ETD data, b and y ions for HCD and b, y, c and z for EThcD. The data were searched with no enzyme specificity and asparagine deamidation and methionine oxidation set as variable modification. Percolator was used to filter the peptide-to-spectrum matches (PSM) to a < 1% false discovery rate (FDR)[29] and the peptide identification list was further filtered for Xcorr score ≥ 1.5. TripleTOF data files were analyzed using identical settings unless otherwise stated. Precursor ion tolerance was set to 20 ppm and the MS/MS tolerance to 0.15 Da. In SEQUEST, spectrum matching was set to 1 for y and b ions. The data were searched with specificity for trypsin and enabling 2 miss cleavages. Oxidation (M), N-terminal acetylation, phosphorylation (S, T and Y), methylation (R, K), dimethylation (R, K) were set as dynamic modification and carbamidomethylation (C) was set as a static modification. The amount of HLA-DR peptides presented at the cell surface expressed as copy number per cell were estimated based on the MS intensities provided by proteome discoverer and known amounts of the synthetic peptides angiotensin-III and oxytocin which were spiked in each fraction prior to LC-MS analysis. CELLO2GO[30] was used for protein subcellular localization prediction. The peptide binding affinities and the 9 a.a. binding core for HLA-DR10, HLA-DR11 and HLA-DR52 were predicted using the NetMHpan-3.0 algorithm[31]. Peptides with a moderate to high binding affinity (IC) < 1000 nM were considered as potential binder for a particular allele. The GibbsCluster-1.0 algorithm[32] was for simultaneous alignment and clustering of complete date set of HLA-DR-associated peptides. Gibbs clustering was performed using default settings[32], with preference of hydrophobic amino acids at position P1, the number of clusters set to 1-4, λ set to 0.8. Sequence logo’s we created using IceLogo[33], with p-values set to 0.005. Linear regression analysis was performed to assess the relation between the abundance data for proteins obtained in the global proteome analysis and in the HLA-DR ligandome analysis of stimulated MUTZ-3 cells, based on log10 transformation of spectral counts-protein length ratio; correlation was expressed as r2.

Contact

Geert Mommen, Intravacc, Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, The Netherlands
Dr Geert Mommen, Intravacc, Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, The Netherlands ( lab head )

Submission Date

21/09/2015

Publication Date

25/01/2016

Cell Type

dendritic cell

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Mommen GP, Marino F, Meiring HD, Poelen MC, van Gaans-van den Brink JA, Mohammed S, Heck AJ, van Els CA. Sampling from the proteome to the HLA-DR ligandome proceeds via high specificity. Mol Cell Proteomics. 2016 Jan 13. pii: mcp.M115.055780 PubMed: 26764012