PROTOCOL

https://doi.org/10.1038/s41596-019-0133-y

Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics

Anthony W. Purcell 1*, Sri H. Ramarathinam 1 and Nicola Ternette2*

Peptide bound to molecules encoded by the major histocompatibility complex (MHC) and presented on the cell surface form the targets of T . This critical arm of the facilitates the eradication of pathogen-infected and cancerous cells, as well as the production of . Methods to identify these peptide antigens are critical to the development of new vaccines, for which the goal is the generation of effective adaptive immune responses and long-lasting immune memory. Here, we describe a robust protocol for the identification of MHC-bound peptides from cell lines and tissues, using nano-ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (nUPLC–MS/MS) and recent improvements in methods for isolation and characterization of these peptides. The protocol starts with the immunoaffinity capture of naturally processed MHC-peptide complexes. The peptides dissociate from the class I human leukocyte antigens (HLAs) upon acid denaturation. This peptide cargo is then extracted and separated into fractions by HPLC, and the peptides in these fractions are identified using nUPLC–MS/MS. With this protocol, several thousand peptides can be identified from a wide variety of cell types, including cancerous and infected cells and those from tissues, with a turnaround time of 2–3d. 1234567890():,; 1234567890():,;

Introduction The recognition of foreign antigens by T lymphocytes is the cornerstone of adaptive . Specifically, intracellular antigens are degraded in the cytosol and antigenic precursor peptides are transported to the endoplasmic reticulum, where they are further trimmed to short peptides (8–11 amino acids (aa)) that can assemble with class I HLAs. These peptide–HLA class I complexes egress + to the cell surface and are scrutinized by CD8 T cells for foreign antigens that differ from the pool of peptides presented in a healthy cellular environment. Identification of a pathogenic, mutated or otherwise distinct will eventually lead to the eradication of the affected cell through the cytotoxic arsenal of these immune effector cells. Alternatively, exogenous antigens can be taken up by professional antigen-presenting cells and degraded in the endosomal compartments of the cell. The resultant peptides (10–20 aa) can be subsequently sampled by class II HLA molecules and presented + on the surface of these cells. Recognition of these complexes by antigen-specific CD4 T cells leads to T-cell activation and the generation of a humoral immune response. This central role played by peptides in immunity has driven efforts to not only identify the antigens that are targeted by immunity but also the HLA-bound peptides that determine the specificity, magnitude and quality of the ensuing immune response. Unlike standard proteomic workflows, in which peptides generated from deliberate proteolytic digestion of proteinaceous samples are identified by mass spectrometry, studies of HLA-bound peptides (immunopeptidomics) are considerably more challenging for a number of reasons: first, the precise proteolytic mechanism for generating the mature peptide–HLA complex is unknown and complex (many proteases and peptidases can be involved in liberating the peptide from the mature antigen complex) (Fig. 1); second, the peptides that bind to HLA molecules are more similar to each other in terms of length and sequence conservation as compared with enzyme-digested protein material; and, finally, the relatively low abundance of HLA-bound peptides, as compared with that of the intact source antigen, makes their detection more difficult1. Thus, historically, the biochemical identification of HLA-bound peptides has been the domain of a few specialized laboratories and, unlike more conventional proteome-based studies, has been restricted to just a few hundred peptides per analysis. In the past 10 years, many of the factors that have hampered HLA immunopeptidome

1Department of Biochemistry and Molecular Biology, Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia. 2The Jenner Institute, Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, Oxford, UK. *e-mail: [email protected]; [email protected]

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Peptide MHC class I pathway Peptide MHC class II pathway

+ CD8 CD4+ T cell

TCR TCR

Exogenous MHC antigen MHC class I class II

Endogenous antigen Endosome

Proteasome

MIIC Peptides Golgi CLIP TAP

Erp57 Crt MHC Tapasin class II Endoplasmic MHC li TAP reticulum β2m class I

Fig. 1 | MHC class I and II antigen-presentation pathways. The peptide MHC class I pathway (blue arrow): endogenous antigens are degraded into peptides by the actions of the cytosolic proteasome. These peptide precursors are subsequently transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) heterodimer. Immature MHC class I molecules are stabilized by tapasin, Erp57 and calreticulin (Crt) (peptide loading complex). Mature MHC class I complexes that are loaded with peptide antigen are transported to the cell surface via the Golgi apparatus. The peptide MHC class II pathway (green arrow): exogenous antigens are taken up via the endosome pathway. Immature class II protein complexes that are stabilized by the invariant chain (Ii) are released into MHC class II compartments via the Golgi apparatus, where the Ii is processed into a short peptide sequence (CLIP) that is then exchanged with the final binding peptide. Mature MHC class II molecules are transported to the cell surface. β2m, β2 microglobulin; MIIC, MHC class II compartment; TCR, T-cell receptor.

studies have been mitigated. This has involved improvements in the efficiency of isolation of HLA- associated peptides and rapid technological developments in instrumentation, bringing greater sen- sitivity, greater resolving power, and much faster acquisition and duty cycles, in addition to sig- nificantly improved computational speed and accuracy of algorithms for searching raw LC–MS/MS data in the absence of protease information. One of the primary roles of antigen processing and presentation resides in the communication of intracellular and extracellular protein expression to the immune system, a process referred to as immune surveillance. By virtue of T scrutiny of endogenous protein expression (the + + CD8 T cell–class I HLA axis) or exogenous (the CD8 T cell–class I HLA axis + for cross-presented antigens and the CD4 T cell–class II HLA axis), the cellular proteome is constantly monitored for changes that have the potential to elicit immune responses (Fig. 1). Recent – studies in which large data sets of HLA-bound peptides have been examined2 14 have started to examine the fidelity of this process and the roles of antigen abundance, protein length, and post- translational modifications, as well as the mechanism of generation of the immunopeptidome. Once identified, these peptide antigens can inform peptide-based vaccine design and can be used as biomarkers in functional assays or incorporated into multimeric reagents for the flow cytometric – detection and enumeration of antigen-specific T cells15 17. Peptide-based vaccines have seen a renaissance in recent years, particularly in the area of universal vaccines that target conserved regions – of pathogens such as HIV, influenza and Plasmodium2,18 23. Their potential for the treatment of cancer has also recently been illuminated by the discovery of neoepitopes associated with many

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cancer types that are targeted by tumor-specific T cells following checkpoint inhibition13,24,25. Tet- rameric peptide–HLA reagents coupled to fluorophores have been broadly used as biomarkers with both diagnostic and prognostic value, as well as for enumerating antigen-specific T cells in a variety of – disease settings21,26 34.

Development of the protocol Several approaches can be taken to isolate HLA-bound peptides from cell lines and tissues. The simplest, yet least specific, approach involves acid stripping the surfaces of cells in culture, using an – isotonic acidic buffer35 37. In our experience, this results in unacceptable levels of contaminating peptides that hamper in-depth analysis of the immunopeptidome. Another approach involves – engineering cells to secrete a soluble HLA molecule of interest38 50. This approach can yield sub- stantial quantities of HLA–peptide complexes that can be easily purified from the medium before nUPLC–MS/MS analysis. It does, however, require the manipulation of the cell line and selection of stable transfectants expressing the soluble HLA molecule of interest, although in general this does not appear to affect the peptide cargo42. It is also not feasible to use this approach to analyze HLA-bound peptides from clinical samples or tissues. We therefore favor an approach that involves direct iso- lation of solubilized HLA–peptide complexes isolated from cell-line or tissue lysates. This approach, – – pioneered by the Engelhard and Hunt51 59, Natheson60 and Rammensee groups61 67, involves the immunoprecipitation of HLA molecules and subsequent dissociation of the bound peptide cargo. A common practice has been to separate these peptides from the heavy chain and, in the case of class I molecules, β2 microglobulin (β2m), using a low-protein-binding MWCO spin filter. In our experi- ence, this results in loss of peptides, irrespective of filter brand and blocking strategies and may also lead to contamination with polymers. Therefore, our protocol describes the separation of the peptides from the larger complex components by using reversed-phase HPLC (RP-HPLC), which, in our experience, increases the peptide yield by up to tenfold. We present here an integrated protocol for the isolation, identification and validation of HLA-bound peptides (Fig. 2) that is based on our – collective experience with this modified protocol6,7,9,68 71.

Level of expertise needed to implement the protocol The protocol detailed here can be followed by most biochemists and immunologists with an eye to detail and experience handling small amounts of material. However, we strongly recommend that an experienced mass spectrometrist be engaged early in the experimental design to facilitate the more demanding aspects of setting up the instrumentation for nUPLC–MS/MS acquisition and subsequent data analysis. Many core facilities are not equipped to do such bespoke analysis and often such sensitive measurements are hampered by contamination from other more typical proteomics samples such as tryptic digests in which peptides may be present at several thousand times higher con- centration than that of peptides isolated from HLA molecules. Thus, great care needs to be taken to ensure the HPLC and mass spectrometer components are both free from any residual carryover material and that the instrument is operating at its absolute highest sensitivity.

Limitations The direct biochemical analysis of HLA-bound peptides, while providing a truly unbiased mea- surement of the nature of the peptide cargo of these molecules, still has limitations. First, the depth of coverage is restricted by the amount of material available for the analysis. For small samples only, the most abundant and most readily detectable (ionizable) peptides will be identified with perhaps only a few hundred peptide sequences determined. In a handful of cases, limitations in detection have been observed that are due to the chemical properties of the : the ability to bind and elute from commonly used desalting media such as C18 during pre-fractionation and subsequent online LC before MS and restricted capability to ionize can lead to impaired detection of the molecule by nUPLC–MS/MS, which requires positive charging in an electrospray source. Finally, suitable anti- bodies are not always available for some HLA alleles or for MHC molecules from other species. For instance, we have recently undertaken a strategy whereby an affinity tag was engineered at the C terminus of a bat MHC class I molecule to facilitate its immunoprecipitation before studying the peptide cargo of this molecule by mass spectrometry72 in order to circumvent the lack of bat- specific reagents.

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Analyte Homogenization

Whole organsCultured cells Biopsies Cryo mill Bead beater Sample preparation

MHC immunoprecipitation HPLC

β2m mAU

MHC-IP Acid MHC elution peptides α-chain MHC-associated Cell lysate MHC MHC peptides peptide purification β complexes 2m α-chain Time

LC–MS/MS Identification FDR

MS2 y8 y7 y6 y5 y4 y3 y2 y1 Random MS

LC b1 b2 b3 b4 b5 b6 b7 b8 y True b2 6 m/z Score m/z No. of spectra y4 y3 5% y5 and data analysis b Decoy Time 3 LC–MS/MS acquisition hits No. of m/z PSMs

Label-free quantification NetMHC prediction Spectral matching and motif analysis Experimental Condition 1 Condition 2

2.0 m/z

Bits 1.0 and validation Further analysis 0.0 Peptide intesity Peptide intesity Time Time 59 Standard WebLogo 3.4

Fig. 2 | Workflow for MHC-associated peptide purification, identification and validation by nUPLC–MS/MS. Top left, MHC complexes are purified from biopsies and tissue or cultured cells. Top right, homogenization of the analyte can be achieved using a cryo mill or bead beater, depending on the type of sample. Second row, left, MHC molecules are purified by immunoprecipitation from the cell lysate, using an specific to the desired MHC species (MHC-IP) followed by acid elution from the resin. Second row, left, MHC-associated peptides are separated from the larger MHC components, α-chain and β2-microglobulin (β2m) by HPLC. Third row, left, the peptide fraction is analyzed by LC tandem mass spectrometry (LC–MS/MS), and (center) peptide sequences are identified by spectral interpretation using de novo–assisted database search engines. Third row, right, FDR (false discovery rate) calculation is performed using decoy databases to control for false-positive identifications and final peptide sequence lists are generated. Bottom left, label-free quantification can be performed between conditions, if applicable. Bottom center, motif analysis and MHC binding prediction of the dataset can be performed as required. Bottom right, spectral matching of the experimental spectra to synthetic peptide standard spectra acquired under identical conditions can be used for validation. PSM, peptide spectrum match.

Experimental design The experimental design typically would involve choosing antigen-presenting cells that express the HLA molecule(s) of interest. Appropriate controls need to be chosen depending on the experiment; for example, for infected or tumor cells, respective healthy cells need to be used, and for HLA or antigen-transfected antigen-presenting cells (APCs), the untransfected parental cell line, or cells containing irrelevant HLA should be used. Although it may not always be possible due to sample limitations, three biological replicates are necessary to draw meaningful conclusions from the data; this is particularly important for studies that compare peptide repertoires between two different states (e.g., mock versus infected cells) or compare the peptide repertoires of related HLA allomorphs.

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A typical experiment may involve identification of HLA–peptide complexes derived from pathogens and evaluation of their using functional assays. Each lab needs to have a good cell line for optimization and quality control (for example, any of the commonly available B lympho- blastoid cell lines that express defined HLA class I and II allomorphs). Moreover, comparing HLA expression of the cells of interest with a quality-controlled cell line can yield valuable insights on the amount of reagents (for example, amount of antibody cross-linked protein A resin) needed for the various steps of the protocol.

Materials

Biological materials ● Tissue, isolated primary cells or cell lines grown in culture ! CAUTION The cell lines used in your research should be regularly checked to ensure that they are authentic and that they are not infected with mycoplasma.

Reagents CRITICAL – c Prepare all solutions using LC MS grade water and use MS-grade solvents and reagents, as well as ultraclean glassware and plasticware. Pre-used and new glassware, as well as plasticware, can leach polymeric substances such as plasticizers and detergent residues that can mask peptide signals during nUPLC–MS/MS analysis. We recommend using high-grade plasticware and always checking tubes and other materials for extractables by LC–MS before using them for this CRITICAL protocol. c For best results, all solutions should be prepared immediately before use and disposed of after 1 week of storage. ● Formic acid (FA, LC–MS Ultra, >98% purity; Sigma-Aldrich, cat. no. 14265-1ML) ● Igepal CA-630 (Sigma-Aldrich, cat. no. I8896) ● Triethanolamine (Thermo Fisher Scientific, cat. no. 787-500ml) ● Pepstatin A (MP Biochemicals, cat. no. 219536810) ● Dimethyl pimelimidate dihydrochloride (DMP-2HCl; Sigma, cat. no. D8388-250MG) ● Complete protease inhibitor cocktail tablets, provided in glass vials (Roche, cat. no. 11836145001) CRITICAL c Use of glass vials results in lower levels of contaminating polymeric material. ● Acetic acid (>99.8% purity, 1 liter in glass bottle; Sigma-Aldrich, cat. no. 33209-1L-GL) CRITICAL c Glass bottles reduce contaminating polymeric material that accumulates during storage. ● Water, Optima LC/MS-grade 4L (Thermo Fisher Scientific, cat. no. FSBW6-4) ● Acetonitrile, Optima LC/MS-grade (Thermo Fisher Scientific, cat. no. FSBA955-4) ● Trifluoroacetic acid (TFA; sequanal-grade, 10 × 1 ml; Thermo Fisher Scientific, cat. no. PIE28904) ! CAUTION TFA is corrosive; appropriate personal protective equipment (PPE) should be worn. CRITICAL Buffers should be prepared in a fume hood due to the risk of toxic emissions. c The 1-ml ampules allow buffers to be freshly prepared and minimize accumulation of contaminants in TFA that can interfere with the analysis. ● Isopropanol (LC–MS Chromasolv; Sigma-Aldrich, cat. no. 34965-2.5L) ● Methanol for LC, LiChrosolv (Merck Millipore, cat. no. 1.06018.4000) ● [Glu1]-Fibrinopeptide B human (≥90% purity by HPLC, 0.1 mg; Sigma-Aldrich, cat. no. F3261) ● Potassium chloride (KCl; Ajax Finechem, cat. no. 383) ● Sodium dihydrogen orthophosphate (NaH2PO4; Ajax Finechem, cat. no. 3964) ● Potassium dihydrogen phosphate (KH2PO4; Ajax Finechem, cat. no. 391) ● Sodium hydroxide (NaOH; Merck Millipore, cat. no. 1.06498.0500) ● Boric acid (Astral Scientific, cat. no. BIOBB0044) ● Citric acid monohydrate (Merck Millipore, cat. no. 1.00244.0500) ● Ethanol, absolute (Ajax Finechem, cat. no. AJA214) ● Ethylenediaminetetraacetic acid disodium salt (EDTA; Chemsupply, cat. no. 9326410003617) ● Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. 472301-100ML) ● SDS–PAGE gel (Mini-PROTEAN TGX 12% Precast Gel, Bio-Rad cat. no. 456-1046) ● Coomassie blue ● Sodium azide (NaN3) ● Liquid nitrogen

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Immunoaffinity purification of MHC class I/class II molecules ● CRITICAL Protein A resin (CaptivA PriMAB 1 L, Repligen, cat. no. CA-PRI-1000) c Check of mAb(s) to be used, as sometimes protein G resin is more appropriate (e.g., for IgG1). ● Tris, Ultra Pure Grade (Astral Scientific, cat. no. BIO3094T) ● Sodium chloride (NaCl; Merck Millipore, cat. no. 1064041000) ● Pepstatin A (MP Biochemicals, cat. no. 219536810) ● Phenylmethylsulfonylfluoride (PMSF; Sigma-Aldrich, cat. no. P7626-1G)

Equipment ● C18 column: 4.6-mm internal diameter × 50- or 100-mm long reversed-phase C18 end-capped HPLC column (Chromolith Speed Rod; Merck, cat. nos. 1514500001 and 1021290001) ● Disposable plastic or glass Econo-Column from Bio-Rad ● Low-protein-binding 1.5-ml tubes for fraction collection (Eppendorf LoBind tubes, cat. no. EPPE0030108.116) ● Autosampler vials for mass spectrometry (Thermo Scientific, cat. no. THC160134) ● LC-MS/MS system (Sciex TripleTof 6600 or Thermo Scientific Fusion Lumos supplemented with nano-flow UPLC) ● Cryo mill (Retsch, model no. MM400) or a bead beater (Bertin Technologies, model no. Precellys 24) ● Microflow HPLC gradient pump system (Ettan HPLC system with an integrated Frac-950 fraction collector (GE Healthcare) or an Ultimate 3000 system supplemented with an AFC 3000 fraction collector (Thermo Scientific)) ● Preparative monolithic RP-HPLC column (Chromolith SpeedROD RP-C18 end-capped, 50 × 4.6-mm column (Merck) or a 50 × 4.6-mm column (Thermo Scientific, model no. ProSwift RP-1S). ● 0.8-μm Filter ● 0.45-μm Filter ● ÄKTAmicro HPLC system (GE Healthcare)

Reagent setup CRITICAL c All solutions for cross-linking, with the exception of triethanolamine and DMP, should be filtered through a 0.2-μM filter and prepared fresh on the day of use.

PBS fi 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4. The sterile- ltered 10× stock solution is stable at room temperature (RT; 22 °C) for 6 months.

Antibody The antibody should be purified and diluted in PBS to 1–10 mg/ml. Ideally, this should represent a CRITICAL single mAb or a mixture of mAbs. c The choice of mAb is closely allied to the choice of the source of HLA–peptide complexes (cell line, tissues and so on) and the specificity and efficacy of the mAbs used in the immunoaffinity isolation of HLA molecules. mAbs with specificity toward classes of MHC molecules, families of MHC molecules, individual alleles of MHC molecules and even subsets of molecules of an individual have been generated over the years, and many hybridomas are readily accessible commercially through bodies such as the ATCC (www.atcc.org). Those we have successfully used for MHC/peptide elution experiments are shown in Table 1, which highlights the fact that some antibodies are better suited for MHC elution than others. For example, although both L243 and LB3.1 antibodies affinity-purify HLA-DR, LB3.1 yields at least double the number of MHC peptides. It is also important to determine whether the DMP cross-linker interferes with the ability of the antibody to immunoprecipitate (in which case other cross-linking methods may be required).

Boric acid–KCl stock solution Prepare 0.1 M boric acid and 0.1 M KCl stock solution by dissolving 0.62 g of boric acid and 0.75 g of KCl in 100 ml of water.

Borate wash buffer For 100 ml, add 3.97 ml of 0.1 N NaOH to 50 ml of boric acid–KCl stock solution and make up the volume to 100 ml with water. Prepare fresh on the day of the experiment.

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Table 1 | Commonly used mAbs for immunoaffinity capture of HLA–peptide complexes

Hybrid Specificity Isotype Ref. ATCC Comments

Anti-human 95 L243 DRα IgG2a Nepom et al. and HB-55 Lower peptide yield than LB3.1 Lampson and Levy96 97 LB3.1 DRα IgG2a Gorga et al. HB-298 Use in preference to L243 95 SPV-L3 DQ IgG2a Nepom et al. 98 IVD12 DQw3 IgG1 Kolstad et al. HB-144 Reactivity to DQw3 alleles 10 IVA12 DR, DQ, DP IgG1 Bassani-Sternberg et al. HB-145 99 B721 DP (DP1-5) IgG3 Watson et al. 100 MA2.1 A2, B17 IgG1 Ellis et al. HB-54 101 BB7.2 A2, A69 IgG2b Parham and Brodsky HB-82 100 ME1 B7, Bw22, B27 IgG1 Ellis et al. HB-119 Cross-reacts with HLA B14 and Bw46 102 W632 A, B, C IgG2a Schittenhelm et al. HB-95 103 DT9 C, E IgG2b Braud et al. and Marginally lower yield of peptides Thomas et al.104 in comparison to W6/32

Protein A wash buffer Protein A wash buffer is 0.2 M triethanolamine, pH 8.2 at RT. Prepare this solution fresh and adjust pH with HCl just before use.

Dimethyl pimelimidate cross-linker Prepare 40 mM DMP-2HCl in 0.2 M triethanolamine, and adjust the pH to 8.3. Prepare DMP by dissolving 250 mg of DMP-2HCl in 22 ml of 0.2 M triethanolamine, and adjust the pH to 8.2. Adjust the pH to 8.3 with NaOH to an approximate final volume of 24.1 ml. Prepare this solution fresh and CRITICAL adjust the pH just before use. c DMP can be bought in larger amounts; however, 250-mg vials work well for single use. This eliminates the possibility of decreased efficiency of cross-linking due to long-term storage of larger quantities of DMP at −20 °C. Generally, one 250-mg vial is used to cross- link up to 20 mg of antibody to 2 ml of resin.

Termination buffer Termination buffer is ice-cold 0.2 M Tris, pH 8.0. Prepare fresh on the day of the experiment from 1 M Tris stock solution.

Citric acid stock solution Citric acid stock solution is 0.5 M citric acid. Prepare by dissolving 105.07 g of citric acid mono- hydrate in 1 L of water.

Stripping buffer Stripping buffer is 0.1 M citrate, pH 3.0. Prepare fresh on the day of the experiment from 0.5 M citric acid stock solution.

Generation of cell or tissue lysate HLA-bound peptides can be affinity-purified from tissue, isolated primary cells or cell lines grown in culture. The yield of MHC class I or class II molecules depends on the tissue or cells used. Epstein- Barr virus–transformed lines that express high levels of HLA-A, HLA-B or HLA-C class I molecules or HLA-DR, HLA-DQ or HLA-DP molecules can be obtained from depositories such as ATCC. These cells can be grown to high density in cell culture and some can be sourced that are homozygous for common class I or class II alleles. If you need to purify a single HLA allomorph, the choice of discriminating antibody depends on which type of APC is used. If there is not an antibody to match this allomorph or to discriminate between the HLA allomorphs expressed by the APC of choice, the ideal cell type for these experi- ments would express a single MHC molecule and have intact antigen-processing and antigen- presentation pathways.

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Several mutant cell lines have been generated that approximate such a cell type: ● The B-lymphoblastoid cell line Hmy2.C1R was generated by gamma irradiation of LICR.LON.Hmy2 (ref. 73) and selected with antibodies against HLA-A and HLA-B alleles and complement. This resulted in a cell line with no detectable HLA-A and very low levels of HLA-B gene products, yet with intact antigen-processing and presentation pathways74. ● The C1R cell line has a defined endogenous peptide repertoire resulting from low levels HLA B*35:03 and normal levels of HLA C*04:01 (ref. 3); moreover these cells are able to support high-level expression of individually transfected HLA-A, HLA-B or HLA-C gene products74. ● Similarly, the 721.221 cell line has an HLA-A-, HLA-B-, HLA-C-null phenotype and can also support the expression of introduced HLA class I molecules75,76. However, this cell line has been shown to have residual HLA-C expression77. ● Class II molecules can also be introduced into mutant APCs such as the human T2 cell line, which has a deletion of the class II loci74,78, and yet supports expression of HLA class II molecules when co- transfected with HLA DM70. ● Likewise, the murine cell line M12.C3 lacks endogenous I-A, and functional I-Ak expression can be restored by introduction of I-Ak α and β chains via transfection79. The number of APCs that can be used for the creation of appropriate cell lines is limited by the fact that not all cells express class II molecules endogenously. The same considerations apply to tissue samples, in which MHC levels may vary and in which there may be a mix of different cell types. The amount of material required is dependent on the depth of analysis anticipated and will be highly dependent on the expression levels of HLA on the cells contained within the sample. For instance, transformed cells grown in culture are the simplest sample type for MHC/peptide isolation, with cell numbers ranging between 5 × 107 and 1 × 109 per isolation. Cells can be expanded and washed in PBS, and the pellets can be snap-frozen in liquid nitrogen for storage at −80 °C for up to 6 months. MHC-associated peptide yields from primary tissue highly depend on the tissue type and amount and type of cells in the analyzed tissue. Generally, 5–200 mg of tissue per experiment is recom- mended. ! CAUTION The cell lines used in your research should be regularly checked to ensure that they are authentic and are not infected with mycoplasma. ! CAUTION The use of animals and tissues derived from them must conform to relevant institutional and national regulations.

1× Lysis buffer 1× Lysis buffer is 0.5% (vol/vol) NP-40 (Igepal CA-630 from Sigma is the equivalent), 50 mM Tris, pH 8.0 (from 1 M stock solution), 150 mM NaCl, and protease inhibitor cocktail (one tablet per 10 ml buffer) (cOmplete Protease Inhibitor from Roche or equivalent, should be made up fresh each time) in Milli-Q H2O. Prepare lysis buffer just before use and keep on ice.

Immunoaffinity purification of MHC class I/class II molecules CRITICAL c We routinely pass the cell lysate (Reagent setup) through an anti-HLA class I column, followed sequentially by columns specific for HLA-DR, then HLA-DQ and finally HLA-DP. ● Pepstatin A: prepare 1 mg/ml stock in isopropanol, divide into aliquots and store at −80 °C (pepstatin A can be stored for at least 6 months at −80 °C). ● Phenylmethylsulfonylfluoride (PMSF): prepare 0.1 M stock in absolute ethanol, divide into aliquots and store at −80 °C (PMSF can be stored for at least 6 months at −80 °C). ● Wash buffer 1 is 0.005% (vol/vol) Igepal CA-630, 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 100 μM PMSF, and 1 μg/ml pepstatin A. Prepare fresh on the day of the experiment. ● Wash buffer 2 is 50 mM Tris, pH 8.0, and 150 mM NaCl. Prepare fresh on the day of the experiment. ● Wash buffer 3 is 50 mM Tris, pH 8.0, 450 mM NaCl. Prepare fresh on the day of the experiment. ● Wash buffer 4 is 50 mM Tris, pH 8.0. Prepare fresh on the day of the experiment. ● Elution buffer is 10% (vol/vol) acetic acid.

Separation of MHC eluate by RP-HPLC ● Buffer A is 0.1% (vol/vol) TFA in MS-grade water. ● CRITICAL Buffer B is 0.1% (vol/vol) TFA in MS-grade acetonitrile. c Alternatively, FA can be used as the ion-pairing agent; although FA is more compatible with mobile phases used in LC–MS, TFA affords better resolution and better complements the FA-based phases of the second dimension of chromatography that is coupled to the LC–MS experiment. Note that TFA suppresses the ESI signal.

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LC–MS/MS analysis of MHC eluate ● Buffer A is 0.1% (vol/vol) FA in MS-grade H2O. ● Buffer B is 0.1% (vol/vol) FA in 80% (vol/vol) MS-grade acetonitrile in water. ● Quality control samples are needed to ensure optimal performance of instruments. ● Instrument-dependent calibration solution for mass spectrometers: we routinely use [Glu1]- fibrinopeptide B to calibrate a SCIEX 5600 TripleTOF mass spectrometer.

Equipment setup Cell pellet/tissue homogenizer For homogenization of large cell pellets and tissue samples, we recommend the use of a cryo mill such as the MM400 mixer mill (Retsch) or a bead beater (e.g., Precellys 24 Bead-Beater; Bertin Technologies).

Pre-column (non-cross-linked protein A sepharose) The bed volume should be half that of the cross-linked column, i.e., for a 1-ml protein A-mAb column, use a 0.5-ml pre-column.

Suitable column Use a disposable plastic or glass Econo-Column from Bio-Rad or equivalent. Alternatively, the CRITICAL immobilized mAb-resin can be packed into small spin columns or stage tips. c Ensure that the column is appropriately washed and soaked in 10% (vol/vol) acetic acid to ensure all leachable material is removed.

RP-HPLC fractionation For RP-HPLC, a standard microflow HPLC gradient pump system with a flow rate capability of up to 500 μl, supplemented with an autosampler, UV detector and fraction collector, is recommended. We use an Ettan HPLC system with an integrated Frac-950 fraction collector (GE Healthcare) or an Ultimate 3000 system supplemented with an AFC 3000 fraction collector (Thermo Scientific). Separation of peptides from the higher-mass components of the HLA complexes is achieved using a preparative monolithic RP-HPLC column. Standard bead-based reversed-phase columns do not exhibit sufficient resolving power for such separation and are more prone to blockage by the protein components of the acid eluate. Columns used in our laboratories are a Chromolith SpeedROD RP- C18 end-capped, 50 × 4.6-mm column (Merck) or a 50 × 4.6-mm ProSwift RP-1S column (Thermo Scientific), which are stored in LC-grade methanol. Details of the gradients and fraction collection can be found in the detailed protocol section.

LC–MS/MS systems For the acquisition of purified HLA-associated peptide preparations, we recommend the use of high- end state-of-the-art LC–MS/MS systems.

LC A high resolving power of the chromatography is critical for separating the complex peptide pre- parations and to achieve a comprehensive view of the immunopeptidome. Peptides are generally loaded onto a trap column using a microflow loading pump at 5–15 μl/min with 0.1% (vol/vol) FA or TFA in water or 2% (vol/vol) ACN. Owing to usually low amounts of peptide material, nano LC is preferred over microflow LC for eluting peptides from a trapping column coupled with an analytical column. Typical gradients elute peptides over 60 or 90 min, ramping from 2 to 25–40% buffer B (0.1% formic acid in ACN) in buffer A (0.1% formic acid in water) and introduced directly into the mass spectrometer using a nanospray source at typical flow rates of 250–300 nl/min. Optionally, buffers can be supplemented with additives such as DMSO (5% (vol/vol) final).

Mass spectrometer Mass spectrometers with high-mass-accuracy analyzers are preferred over the use of low-mass-accuracy analyzers such as ion traps for both MS and MS/MS. High mass accuracy can improve the reliability of peptide sequence identification, particularly if software using combined de novo and precursor-based approaches is used for identification. We encourage the use of high targets and long accumulation times for MS2 spectra for an improved spectral quality and spectral yields of up to 30–40%. Accumulation time of 100–120 ms for fragment spectra acquisition improves the quality of spectra recorded and

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Table 2 | Instrument-specific settings for SCIEX and Thermo Scientific mass spectrometers

SCIEX MS1 TOF range 200–1,800 m/z Ion spray voltage (ISVF) 2,400 V Curtain gas 25 liters per min Ion source gas 1 (GS1) 10 liters per min Interface heater temperature (IHT) 150 °C Precursors selected for MS/MS 20 per cycle Charge state 2+ to 5+ Intensity threshold for MS/MS >40 counts per s MS/MS acquisition time 100 ms Collision energy Rolling CE Calibration [Glu1]-fibrinopeptide B Q-Exactive and Q-Exactive HF Spray voltage 1,700–2,200 V Capillary temperature 275 °C Full-scan MS range 300–1,800 m/z MS1 resolution (m/z 200) 70,000 Ion target 5.00 × 105 Maximum injection time 120 ms Precursors selected for MS/MS Top 20 per cycle Precursor charge state 2–4+ Higher-energy collisional dissociation (HCD) 27% MS2 accumulation time 120 ms MS/MS resolution (Orbitrap) 35,000 Target 1.00 × 105 Isolation width 1.2 Da Underfill ratio 1% Dynamic exclusion 15 s Thermo Fusion and Fusion Lumos (differing from Q-Exactive settings) MS1 resolution 120,000 Precursor selection Top-speed mode with 2-s cycle time Precursor charge state 1+;2+ to 4+ (priority) HCD 28% (charge state 2–4); 32 for charge 1 MS/MS resolution (Orbitrap) 60,000 Dynamic exclusion 60 s

maximizes the number of identified spectra in the data set. For Orbitrap instruments, it is critical to set the ion target high in order to avoid limiting the maximal injection time. Note that the acquisition of singly charged precursors for MHC class I preparation can increase the numbers of peptide identifi- cations substantially, depending on the MHC allele combination in the sample. A high number of singly charged peptides can occur in samples in which the HLA allele does not contain basic amino acid residues that can accept a positive charge. Singly charged precursor selection is ideally set to frag- mentation at higher collision energy and at lower priority, as singly charged contaminants may otherwise be prioritized over multiply charged peptide ion precursors. Such settings are only available in Orbitrap Fusion instruments. Suggested settings for SCIEX TripleTOF, Thermo ScientificQ-Exac- tive, Q-Exactive-HF, Fusion and Fusion Lumos LC–MS are detailed in Table 2.

Software The large-scale data acquired from high-resolution mass spectrometers are interpreted using algorithms that enable assignment of mass spectra to amino acid sequences. There is a variety of software available to the community for interpretation of MS fragment spectra, which ranges from commercial software to open-source algorithms in varying stages of development (including MASCOT80, SEQUEST81,X! Tandem82, Maxquant83 and Protein Pilot84). These algorithms rely on an input database of proteins to generate a search space within which the peptide identifications will be confined.

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1 2 3 5 6 Preparation of Antibody Removal of free 4 Termination Stripping of 7 protein A sepharose binding residual amines Cross-linking of cross-linking unbound antibody Equilibration 1 ml of 1–10 mg of 10 CVs protein A antibody 10 CVs 5 CVs 0.2 M Tris 10 CVs 10 CVs sepharose in PBS borate buffer 40 mM DMP (ice-cold) citrate buffer PBS

10 CV 0.2 M TEO

30 min 1 h Fig. 3 | Generation of MHC immunoaffinity column. The first seven steps of the Procedure are shown. CV, column volume; DMP, dimethyl pimelimidate dihydrochloride; TEO, triethanolamine.

There is, however, de novo–based software that utilizes both de novo and database-driven approaches (e.g., PEAKS85). Such software uses preliminary information from de novo searches in order to limit the database search space for a classic precursor mass database search and can thereby improve the speed and the performance of database search engines and is hence recommended for MHC-associated peptide identification. Note that the majority of software has been developed for standard bottom-up proteomics in which full-length proteins are digested with trypsin to produce peptides of a length that is optimal for measurement by standard LC–MS/MS. For tryptic peptides, precursor-based search engines have been shown to be very effective. Only recently, in order to achieve improved protein coverage, the panel of enzymes used in standard MS experiments has been expanded86,87. Particularly for peptides that were generated by multiple enzymes or enzymes with no particular specificity, and for MHC- associated peptides, it is advisable to use algorithms that include a de novo–based approach such as ProteinPilot (v.5; https://sciex.com/products/software/proteinpilot-software) and PEAKS (v.8.5; http://www.bioinfor.com/peaks-studio/).

Computational hardware Irrespective of the search strategy chosen, it is a computationally intensive task and it is recom- mended to have access to powerful computers: 16- to 32-core processors and at least 64 GB of RAM are necessary for reliable operation. If the search algorithm supports multi-threaded multi-core processors, it is preferable; however, not all search algorithms are compatible at this stage.

Procedure Generation of MHC immunoaffinity column ● Timing 2 h 30 min CRITICAL fi c Refer to Fig. 3 for a schematic of the rst section of the Procedure. 1 Preparation of protein A sepharose. Transfer the required amount of protein A sepharose (supplied as a 50% (wt/vol) slurry in 20% (vol/vol) ethanol) to a 50-ml Falcon tube and exchange the buffer to PBS by centrifugation (RT, 4,000g, pulse spin only required). 2 Antibody binding. Prepare the mAb ideally at 0.5–1 mg/ml in PBS. Add the antibody to the protein A sepharose resin. Rotate gently end-over-end at room temperature for 30 min to allow binding. For a typical experiment using 5 × 108 cells, a 1-ml bed volume should be used. CRITICAL STEP c Before cross-linking, it is important to check the quality of the antibody batch by evaluating its binding characteristics using flow cytometry. This staining experiment should be done using cells that express the HLA of choice. For each new antibody, also check the efficiency of the cross-linking reaction by SDS–PAGE. Take a sample of the original antibody (1) and a sample of the flow-through following incubation with the resin (2). Take 25-μl aliquots of resin before the addition of DMP (3) and after incubation with DMP (4). Sample (5) is generated by concentrating the flow-through from the citric acid wash (5) to ~500 μl, using a 15–30 kDa cutoff concentrator (this is done to see how much antibody has been left unbound); take 25 μl to add to 2× SDS–PAGE loading dye. Run the five samples on a reducing SDS–PAGE gel and stain with Coomassie blue. Heavy and light chains of the antibody should be seen in samples 1 and 3. There should be little antibody in samples 2, 4 and 5. If the antibody has not been used for DMP cross-linking before, perform a small-scale immunoprecipitation to test that the antibody still retains its binding specificity. ? TROUBLESHOOTING

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8 and 9 10 11 12 13 14 Lysis and Lysate Protein A sepharose MHC Wash Elution homogenization clearing pre-clearing immunoprecipitation of the lysate 2x Lysis buffer Centrifugation 1 10 CVs 5 CVs at 2,000g, 4 °C wash buffer 10% acetic 10 min Cleared lysate Cleared lysate 1–4 acid Cells

Pipetting A Centrifugation 2 2x Lysis buffer at 100,000g, 4 °C 75 min >1 h

Bead beater Organs/tissue or cryo mill Fig. 4 | MHC immunoaffinity purification. Steps 8–14 of the Procedure are shown.

3 Removal of residual free amines. Transfer the resin and antibody back to the column and allow the antibody to flow through by gravity. Wash the antibody-bound resin with 10 c.v. (10 column volumes) of borate wash buffer, followed by 10 c.v. of freshly prepared 0.2 M triethanolamine, pH 8.2, at RT. CRITICAL STEP c This step is necessary to ensure there are no residual primary amines present that may interfere with the cross-linking reaction. 4 Cross-linking. Flow 5 c.v. of freshly prepared DMP cross-linker through the column at RT, leaving a meniscus just over the protein A column bed. Seal the bottom of column and allow the reaction to proceed at RT for 1 h. ! CAUTION The DMP solution is toxic and PPE should be worn during these steps. 5 Termination of cross-linking. Terminate the cross-linking reaction by adding 10 c.v. of ice-cold termination buffer (0.2 M Tris, pH 8.0). 6 Stripping of unbound antibody. Remove unbound antibody by washing with 10 c.v of stripping buffer (0.1 M citrate, pH 3). 7 Equilibration. Flow 10 c.v. of PBS, pH 7.4, until the pH of the flow-through is >7. PAUSE POINT j It may be convenient to stop here, and wash and store the column at 4 °C in PBS, pH 7.4, supplemented with 0.02% (wt/vol) NaN3. Generally, columns are best used within a month; however, this will vary depending on the specific antibody.

Generation of cell lysate and MHC immunoaffinity purification ● Timing 3 h 30 min CRITICAL – c Refer to Fig. 4 for a schematic showing Steps 8 14. 8 Preservation of cells for immunoaffinity purification. Wash cells (5 × 107 to 1 × 109) with PBS and harvest by centrifugation (2,000g, 10 min at 4 °C). Pellets can be snap-frozen in liquid nitrogen and can be stored at −80 °C for up to 6 months. If processing tissues or biopsies, a range of 2–200 mg of tissue can be processed. Snap-freeze the tissue upon collection and store at −80 °C until processing. ! CAUTION Liquid nitrogen poses an asphyxiation hazard, and skin contact can result in cold burns; wear PPE and handle the material in a well-ventilated area. CRITICAL STEP c It is important that tissues be frozen upon collection, to ensure the integrity of the HLA–peptide complexes on the cells. 9 Lysis and homogenization. For large cell pellets or tissue samples (mouse spleens and lymph nodes), we recommend grinding cells under liquid nitrogen or using a bead beater. For cell grinding, place the snap-frozen pellets straight into a Mixer Mill MM 400, and, after grinding, scrape the powder into lysis buffer. Alternatively, tissue samples in lysis buffer can be processed using a Precellys 24 Bead-Beater. To do this, use at least five 15-s cycles at the highest speed setting (6500). Proceed as described for cell lysis. Small cell pellets can be homogenized in lysis buffer by pipetting up and down. 10 Lysate clearing. Centrifuge the lysate for 10 min at 2,000g (4 °C). This step removes the nuclei. Take the supernatant and spin it for 75 min in an ultracentrifuge (100,000g) at 4 °C. Collect the supernatant, it should appear clear. If there is an unclear layer at top of the tubes, carefully remove this lipid-containing layer and filter it through a 0.8-μm filter and then a 0.45-μm filter. This step is also required when working with bacteria or parasites in order to remove infectious units from the lysate.

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11 Pre-clearing of the lysate. Using gravity flow or a peristaltic pump in a cold room, load the cell lysate onto a protein A sepharose pre-column that has been pre-equilibrated in 10 c.v. of wash buffer 1. Multiple pre-columns may be required, depending on the size and type of sample and should be replaced upon any clogging. 12 MHC immunoprecipitation. Collect pre-cleared lysate and slowly load it onto the cross-linked mAb column(s). If the gravity flow is too quick (<1 h for lysate to pass), use a peristaltic pump. For small samples (1–4 ml of lysate), it is generally better to add lysate to several 2-ml LoBind Eppendorf tubes containing resin and rotate them slowly end-over-end at 4 °C for 1 h. For maximal yield, the lysate should be run through the column twice. If desired, the flow-through can be retained and frozen at −80 °C for subsequent proteome analysis. If multiple columns are being run (i.e., class I and II elutions from the same lysate), we routinely run the lysate once only over each column. The columns can be set up on a retort stand in parallel so that the flow-through from one column runs directly into the next. It may be necessary to temporarily cap different columns throughout the process, to ensure they do not dry out (if the lysate is passing at slightly different flow rates through each). If binding has been performed in 2-ml LoBind Eppendorf tubes, after the 1-h incubation, spin the resin for 1 min at 2,000g (4 °C) and transfer the supernatant to an Eppendorf tube containing the next mAb resin. Repeat the incubation while washing and eluting from the first batch of mAb resin. 13 Washing. Wash the column(s) with 10 c.v. of each wash buffer in the following order: wash buffer 1, wash buffer 2 (to remove detergent), wash buffer 3, (to remove non-specifically bound material), wash buffer 4 (to remove salt to prevent crystal formation). 14 Elution. Elute MHC molecules in 5 c.v. of elution buffer. We recommend that you perform RP-HPLC fractionation of the eluate immediately. PAUSE POINT − j Alternatively, the eluate can be frozen at 80 °C; however, this may result in some sample loss.

Separation of MHC eluate by RP-HPLC ● Timing 1 h per sample CRITICAL c A single RP-HPLC step can be used to enrich the peptides if the fractions will be analyzed using an immunological assay. For mass spectrometry, however, it is necessary to perform at least two dimensions of RP-HPLC to produce peptides that are sufficiently separated for analysis. The first dimension could be an offline RP-HPLC in which fractions are collected and pooled in a meaningful way before LC–MS analysis. This step also serves to remove the heavy chain and β2m, as well as leached antibody, from HLA peptides. 15 Enrich the peptides (separate them from the MHC heavy chain, β2m (for class I molecules), leached antibody and some forms of contaminating detergent) using a C18 reversed-phase column running on a mobile phase buffer A of 0.1% TFA and buffer B of 80% acetonitrile (ACN)/0.1% TFA. We routinely use a 4.6-mm internal diameter × 50-mm long reversed-phase C18 end-capped HPLC column (Chromolith Speed Rod) on an ÄKTAmicro HPLC system. Separate based on a rapid gradient of buffer A to B, which results in 10–30 peptide-containing fractions (e.g., 2–40% B for 4 min, 40–45% for 4 min, and a rapid 2-min increase to 100% B; Fig. 2). Using this approach, a small number of early fractions contain >95% of the peptides, whereas the later fractions contain β2-microglobulin, HLA heavy chains, and, additionally, Igepal CA-630 polymers, which severely hamper MS analysis. Look at the area of the β2m and the heavy-chain peaks in the UV trace; this provides vital information pertaining to the efficiency of MHC elution. CRITICAL STEP c The number of fractions collected needs to be a compromise between reducing the complexity of the sample for mass spectrometric analysis and keeping the number of samples to a practical number. We routinely collect 500 μl of fractions, then pool these into a single concatenated sample consisting of four to six fractions spread well across the gradient separation. For example, to obtain six combined pools, sample 1 would be combined with samples 7 and 13. In this way, the number of samples for LC–MS/MS is reduced, and the full gradient on the column attached to the mass spectrometer is utilized. Caution should be exercised with fractions coming off in high ACN; although these may contain peptides of interest, the highly hydrophobic nature of the peptides and proteinaceous material in these samples may interfere with detection of more hydrophilic peptides due to displacement of these species from trap columns. If these fractions contain a lot of material, on the basis of UV intensity, it may be better to run these as individual fractions.

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16 Collect the fractions (500 μl) into LoBind Eppendorf tubes. At this point, fractions can be frozen at −80 °C for up to 1 year. ? TROUBLESHOOTING 17 Vacuum-concentrate the peptide-containing fractions (generally, those fractions eluted in up to 42% buffer B) to reduce the concentration of ACN. Typically, dry to 10 μl and dilute to 15–25 μlin 0.1% formic acid. CRITICAL STEP c Do not dry to completeness, as this may result in sample loss due to adsorption to the plasticware. PAUSE POINT − j Peptides could be frozen and stored at 80 °C until analysis on a mass spectrometer, although it is preferable to proceed directly to the next step.

LC–MS/MS analysis of MHC eluate ● Timing 8–16+ h CRITICAL fi c Numerous factors can affect the resulting number of peptide identi cations from a global LC–MS/MS analysis, including initial starting cell number, expression level of the MHC molecules at the cell surface, efficiency of cell lysis, quantity of antibody used for immunoaffinity capture, appropriate and sufficient upstream HPLC fractionation, on-line LC gradient and specific MS parameter settings. It is recommended to optimize these variables. 18 Place the samples in a sonicating water bath for 5 min to detach the peptides bound to the plastic. CRITICAL STEP c It is important to ensure that the peptides are completely resuspended to prevent loss. 19 Centrifuge the sample at 13,000g for 10 min at 4 °C to pellet any particulates and transfer the supernatant to an autosampler vial. CRITICAL STEP c Failure to remove any particulate matter could lead to blockages of the downstream nanoLC systems. ? TROUBLESHOOTING 20 Load the sample onto the mass spectrometer and run an optimized gradient as described in Equipment setup. CRITICAL STEP c The parameters used for data acquisition need to be carefully considered and operation of the instrument performed by an experienced mass spectrometrist. The decision to examine +1-charge-state peptides, the number of ions selected per cycle and the accumulation time need to be tailored to the analysis to achieve the most information-rich MS/MS spectra. An example of MS/MS spectra for a class I bound peptide is shown in Fig. 5d. ? TROUBLESHOOTING

Data analysis ● Timing 2–16+ h 21 Search. Search the raw data with your favorite search engine and/or protein database. We routinely search ProteinPilot (v.5) or PEAKS (v.8.5) using the parameters for data acquisition mentioned in Step 20 for the identification of HLA peptides. Strict bioinformatic criteria need to be applied to ensure that only the best hits are chosen for further analysis. Use a decoy database to control the false-discovery rate (FDR). An FDR cutoff of either 1 or 5% could be applied. Given that the majority of search engines are geared toward a protein-centric identification approach, they sometimes penalize proteins that are represented by only one peptide, which is often the case for many HLA peptide repertoires88. ? TROUBLESHOOTING 22 Data curation, length, motif analysis and MHC-binding prediction. Carefully evaluate the HLA- bound peptide elution data to ensure the quality of the acquired data. Prediction tools for evaluation of the obtained experimental peptide data are available from resources such as the Immune Epitope Database and Analysis Resource (IEDB; http://www.iedb.org/) and NetMHC 4.0 (refs. 89,90; http://www.cbs.dtu.dk/services/NetMHC/). 23 (Optional) Plot the length distribution of HLA class I and class II peptides as a frequency distribution to provide further confirmation of successful elution of HLA–peptide complexes (see Anticipated results; Fig. 5e). 24 Remove known contaminant peptides (peptides identified from the pre-column and other lab contaminant peptides cataloged over many experimental controls) from the final datasets to ensure high-quality peptide repertoire data.

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a Fraction d B10

2,500 b1 1.2 × 105 582.3

1.1 × 105 1,500

6.0 × 104 β Heavy Intensity 2m b8 chain y2 y8 Absorbance (mAU) 3 500 4.0 × 10 b6 y9 y3 b4 b7 Peptides Buffer B % b2 y4 b5 y6 0 UV 215 nm A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 B15 B14 B13 B12 B11 B10B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10C11C12C13C14C15D15D14 D13D12D11D10 Waste 0 0 10 20 30 200 400 600 800 1,000 b RT (min)(min) e m/z 40 8.0 × 107

30 6.0 × 107

4.0 × 107 20 Intensity % Peptides

2.0 × 107 10

0 0 10 20 30 40 50 60 70 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 c RTT (min)((mi)in) f Length 5 1.2 × 10 582.3 67

9.0 × 104 33

6.0 × 104 0 123456789 Intensity

3.0 × 104 % Difference –33

0 300 400 500 600 700 800 900 1,000 1,100 –67 m/z Fig. 5 | Overview of anticipated results. a, Following reversed-phase chromatography, peptide-containing fractions were collected and subjected to centrifugal evaporation and individually analyzed on the mass spectrometer to reduce complexity of the sample. b, A representative total ion chromatogram (TIC) of one of the fractions (B10). c, The ions detected by the mass spectrometer are subjected to a survey scan, from which the top 20 peaks are chosen for MS/MS analysis in any given cycle. d, MS/MS spectra of one of the peptides (represented by orange circle in c) that was fragmented and subsequently identified using ProteinPilot software. e, The frequency distribution of the peptide lengths of class I peptides (blue bars), which predominantly are 9-mers, in contrast to class II peptides (orange bars), which are longer on average. Data shown are representative of at least three independent experiments and the error bars represent mean ± s.e.m. f, The peptide-binding motif of HLA-A*02:01 was constructed on the basis of the nonamer peptides, using Icelogo (n = 20,000).

25 (Optional) Visualize the immunopeptidomic datasets. This can be easily achieved with online tools such as WebLogo 3 (ref. 91; http://weblogo.threeplusone.com) or Seq2Logo92 (http://www.cbs.dtu. dk/biotools/Seq2Logo/). 26 (Optional) Use Gibbs algorithm93 and/or other clustering approaches94 to perform unsupervised clustering of HLA peptides; as the HLA peptides cluster together, this aids in effective removal of contaminating peptides.

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Troubleshooting Troubleshooting advice can be found in Table 3.

Table 3 | Troubleshooting table

Step Problem Possible reason Solution

Step 2 Low yield of HLA Antibody not efficient at Check each batch of antibody by flow cytometry to complexes/low number of recognizing HLA ensure it binds to the correct MHC peptides identified Antibody did not cross-link effectively Ensure cross-linking was effective. Check the pH of buffers used Check antibody by running SDS–PAGE Step 16 Low yield of HLA Poor capture of MHC–peptide Ensure that HLA–peptide complexes were eluted complexes/low number of complexes; failure to efficiently elute properly. This can be verified by the area under peptides identified complexes the β2-microglobulin and heavy-chain peaks (UV 215-nm trace) during the initial reversed-phase chromatography step. See Fig. 5a. Check the pH of all the buffers used Steps Low number of peptides Sample lost during fractionation and Ensure that samples are not completely dried during 19 and 20 identified concentration centrifugal evaporation, and that they are resuspended carefully. This avoids loss of material due to binding to the tube surface Step 21 Low number of peptides Mass spectrometer is not performing Ensure that the mass spectrometer is functioning to identified optimally specifications. Check this by regularly tuning the instrument and verify by running quality-control samples (e.g., an HLA–peptide complex preparation from a cell line containing a defined number of detectable peptides) at regular intervals

Table 4 | Anticipated yield (varies by cell type and HLA expression)

Number of peptide sequences

Jurkat cells 1×107 100 1×108 3,000 1×109 8,000 Tissue biopsies 2mm3 (2 mg) 100 20 mm3 (20 mg) 1,500–3,000 200 mm3 (200 mg) >10,000

Timing Steps 1–7, generation of MHC immunoaffinity column: 2 h 30 min Steps 8–14, immunoaffinity purification of HLA–peptide complexes: 3 h 30 min Steps 15–17, reversed-phase separation of HLA eluate: 1 h per sample Steps 18–20, LC/MS analysis of HLA eluate: 8–16+ h Steps 21–26, data analysis: 2–16+ h (depending on complexity of sample and depth of analysis)

Anticipated results The yield of HLA-bound peptides depends on the number of cells used (Table 4) and their respective HLA expressions. The immunopeptidomics approach was applied to identify naturally processed ligands from C1R cells transfected with HLA-A*02:01. A total of 1 × 109 cells were lysed using 2× lysis buffer, followed

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by centrifugation to remove nuclei. The clarified lysate was loaded onto an affinity column composed of 10 mg of w6/32 antibody cross-linked to protein A resin. The affinity-purified HLA–peptide complexes were eluted by addition of 10% (vol/vol) acetic acid and separated using reversed-phase chromatography, and fractions collected (Fig. 5a) were analyzed by mass spectrometry (Fig. 5b–f). The data acquired were searched against the human proteome (Uniprot/Swissprot v.2014_10) using ProteinPilot software (v.4.5). After application of the 5% FDR and 0.05 delta mass cutoff values and removal of matches to reversed protein (decoy) hits and common contaminants, 3,724 peptides were identified. As described elsewhere in detail42, the majority of the peptides identified were 9-, 10- or 11-mers, and the analysis of the HLA motif matched the previously described motif with a strong preference for L at position 2 and for V or L at the C terminus (P9 or Ω).

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Acknowledgements We thank the following funding sources for supporting this work: Australian National Health and Medical Research Council (NHMRC) project grants APP1085018 and APP1084283, and Australian Research Council (ARC) project grant DP150104503. A.W.P. was sup- ported by an NHMRC Principal Research Fellowship (APP 1137739).

Author contributions A.W.P., S.H.R. and N.T. wrote the manuscript and collated data and figures.

Competing interests The authors declare no competing interests.

Additional information Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to A.W.P. or N.T. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 18 August 2016; Accepted: 8 January 2019; Published online: 15 May 2019

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Related links Key reference(s) using this protocol Schittenhelm, R. B., Dudek, N. L., Croft, N. P., Ramarathinam, S. H. & Purcell, A. W. Mol. Cell. Proteomics 15, 1867–1876 (2016): https://doi.org/10.1074/mcp.M115.056358 Scally, S. W. et al. J. Exp. Med. 210, 2569–2582 (2013): https://doi.org/10.1084/jem.20131241 Illing, P. T. et al. Nature 486, 554–558 (2012): https://doi.org/10.1038/nature11147 Wynne, J. W. et al. J. Immunol. 196, 4468–4476 (2016): https://doi.org/10.4049/jimmunol.1502062

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