Cross-linking of the human MHC-I peptide-loading complex
The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum (ER), essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the ER with loading and editing of major histocompatibility complex class I molecules (MHC-I). After final proofreading in the PLC, stable peptide/MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits into a single macromolecular assembly, including the transporter associated with antigen processing TAP1/2, the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization and mechanistic events in the PLC are unknown due to the compositional heterogeneity and intrinsic dynamic nature of the complex. Here, PLC from Burkitt’s lymphoma cells was isolated using an engineered viral inhibitor as bait, followed by cross-linking mass spectrometry to aid EM structure elucidation.
Sample Processing Protocol
10 to 30 µg PLC were incubated with varying amounts of bis(sulfosuccinimidyl)suberate (BS3) for one hour at 25 °C. After cross-linking, the proteins were separated by gel electrophoresis or precipitated with ethanol followed by digestion in-solution. Gel electrophoresis and in-gel digestion Proteins were separated by gel electrophoresis using the NuPAGE system (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteins were stained with InstantBlue Coomassie staining solution (expedeon) and protein bands of interest were cut. Proteins were where subject ot tryptic in-gel digestion. In-solution digestion Proteins were precipitated with 100 % (v/v) ethanol. The pellet was washed with 80 % (v/v) ethanol and dried in a vacuum centrifuge. The pellet was dissolved in 1 % (m/v) RapiGest surfactant (Waters) in 25 mM ammonium bicarbonate according to the manufacturer’s protocol. Briefly, disulfide bridges were reduced with 50 mM dithiothreitol and cysteine residues were alkylated with 100 mM iodoacet amide. The proteins were digested with Trypsin at 37 °C overnight in0.1 % (m/v) RapiGest. Peptides were dried in a vacuum centrifuge. LC-MS/MS analysis Peptides were dissolved in 2 % (v/v) acetonitrile (ACN) / 0.1 % (v/v) formic acid (FA) and separated by nanoflow-liquid chromatography on an DionexUltiMate 3000 RSLC nano System (Thermo Scientific) coupled with a Q Exactive plus hybrid mass spectrometer (Thermo Scientific) using mobile phase A, 0.1% (v/v) FA; mobile phase B, 80 % (v/v) ACN /0.1% (v/v) FA. The peptides were loaded onto a precolumn (HPLC column Acclaim® PepMap 100, C18, 100 μm I.D. particle size 5 μm; Thermo scientific) and separated on an analytical column (50 cm, HPLC column Acclaim® PepMap 100, C18, 75 μm I.D. particle size 3 μm; Thermo Scientific) at a flow rate of 300 nl/min with a gradient of 4-90% solvent B over 95 mins. Peptides were directly eluted into the Q Exactive plus hybrid mass spectrometer (Thermo Scientific). MS conditions were: spray voltage of 2.0 kV; capillary temperature of 275 °C; normalized collision energy 30. The Q Exactive was operated in data-dependent mode. MS spectra were acquired in the orbitrap (m/z 350−1600) with a resolution of 70,000 at m/z 400 and an automatic gain control target of 3×10e6. The ten most intense ions were selected for HCD fragmentation at an automatic gain control target of 17,500. Previously selected ions were dynamically excluded for 30 s. Singly charged ions as well as ions with unrecognized charge state were also excluded. Internal calibration of the orbitrap was performed using the lock mass option. For cross-linking samples, the mass tags option was employed, i.e. only ions from peak pairs with mass difference of 4 Da were selected for HCD fragmentation. If no peak pairs were present other ions were selected. In addition, doubly charged ions were excluded from fragmentation.
Data Processing Protocol
Raw files were converted into mgf’s using pXtract (http://pfind.ict.ac.cn/software/pXtract/index.html). For protein identification, mgf’s were searched against SwissProt database (550,299 sequences) using Mascot search engine 188.8.131.52 (Matrix Science). Search parameters were: Taxonomy, Homo Sapiens; peptide mass tolerance, 10 ppm; fragment mass tolerance, 0.5 Da; enzyme, trypsin; variable modifications, carbamidomethylation (cysteine) and oxidation (methionine). For identification of cross-linked peptide, mgf’s were searched against a reduced database containing sequences of the peptide loading complex using pLink software. Search parameters were: instrument spectra, HCD; enzyme, trypsin; max. missed cleavage sites, 4; variable modifications, oxidation (methionine) and carbamidomethylation (cysteine); cross-linker, BS3 (lysine-lysine)/BS3d4 (lysine-lysine); min. peptide length, 4; max. peptide length, 100; min. peptide mass, 400 Da; max. peptide mass, 10,000 Da; FRD, 1%. Potential cross-linked di-peptides was evaluated by their spectral quality.
Tommy Hofmann, Interdisciplinary research center HALOmem, Martin Luther University Halle-Wittenberg, Kurt-Moses-Straße-3, 06120 Halle (Saale
Carla Schmidt, Interdisciplinary research center HALOmem, Martin Luther University Halle-Wittenberg, Kurt-Moses-Straße-3, 06120 Halle (Saale) ( lab head )
Blees A, Januliene D, Hofmann T, Koller N, Schmidt C, Trowitzsch S, Moeller A, Tampé R. Structure of the human MHC-I peptide-loading complex. Nature. 2017 PubMed: 29107940