C18 spin column

EV pellets were solubilised in 8 m urea/50 mM NH4HCO3/0.1% ProteaseMax using sonication, with resultant protein levels quantified using a Bradford assay. Following dithiothreitol reduction and iodoacetic acid-mediated alkylation, a double digestion was performed using Lys-C (for 4 h at 37 °C) and Trypsin (overnight at 37 °C) on 5 μg of protein. The samples were desalted prior to analysis using C18 spin columns (Thermo Scientific) and 500ng was loaded onto an Ultimate 3000 NanoLC system (Dionex Corporation, Sunnyvale, CA, USA) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The raw data were analysed using Progenesis QI for Proteomics software (version 3.1; Non-Linear Dynamics, a Waters company, Newcastle upon Tyne, UK) as previously described,^{61 (link)} with following modifications: the filter mass peaks with charge states from+1 to+6 was applied to the MS/MS data files, peptides were identified using taxonomy: Homo sapiens in the SwissProt database and peptides with XCorr scores>1.9 for +1 ions,>2.2 for +2 ions and>3.75 for +3 ions or more (from Sequest HT) were selected.

Peptide separation was performed using a Polysulfoethyl A Column (200 mm L × 2.1 mm i.d., 5 μm, 300 Å, PolyLC, Columbia, MD) with an Agilent 1100 binary HPLC (Agilent Technologies, Wilmington, DE) over a 90 min gradient. Eighty fractions were collected after SCX fractionation and then pooled into 24 fractions. The fractions were purified using C-18 Spin columns (Thermo Scientific, San Jose, CA) for further nano-LC-ESI-MS/MS analysis14 (link).

Ubiquitination reaction samples (3 µg protein) were diluted in denaturing buffer (3 M urea, 25 mM TEAB pH 8.5, 0.5 mM EDTA) and reduced using 2 mM TCEP for 10 min at 37°C, followed by alkylation with 50 mM chloroacetamide for 30 min at room temperature in the dark. Chloroacetamide was used to avoid iodoacetamide-induced artefacts that mimic ubiquitination [84 (link)]. Samples were diluted with 50 mM TEAB pH 8.5 to 1 M urea and digested with 0.5 µg trypsin (Sigma) for 3 h at 37°C. Digested peptides were purified using C18 Spin Columns (ThermoFisher) and resuspended in 0.1% formic acid. Peptides (0.5 µg) were captured and eluted from an Acclaim PepMap100 C18 column with a 2 h gradient of acetonitrile in 0.1% formic acid at 200 ml min⁻¹. The eluted peptides were analysed with an Impact II Q-TOF spectrometer equipped with a Captive Spray nanoelectrospray source (Bruker). Data were acquired using data-dependent automatic tandem mass spectrometry (auto-MS/MS) and analysed with MaxQuant using a standard search procedure against a custom-made FASTA file including GST-Ub, Parkin, Ub and UbcH7. Methionine oxidation, Lys ubiquitination (diGly) and Ser/Thr phosphorylation were included as variable modifications. Cysteine carbamylation was included as fixed modification.

Cell culture and dosing conditions were the same as metabolomics methods. The proteins of cells were extracted and quantified. Then equal proteins (approximately 30 µg) were desalted and digested. The peptides mixture were desalted using C₁₈ spin columns (Product # 89870, Thermo Scientific) and the flow-through was dried in a SpeedVac centrifugal evaporator. The dried peptides were dissolved in water for 2D LC-MS/MS analysis. A Waters system (Waters, Milford, MA) of 2D nano-Acquity UPLC coupled with Q-TOF premier tandem mass spectrometer was used for proteomics studies. Raw data from 2D nano-UPLC/Q-TOF were analyzed by the ProteinLynx Global Server (PLGS 2.5) with ExpressionE informatics. A Swiss-Prot database (release 51.0, October 2013) was used for database searches.

We followed our original procedure (Wan et al., 2020 (link)), with modifications. The WBCs were suspended in lysis buffer (40 mM N-octanoyl-N-methylglucamine, 40 mM N-nonanoyl-N-methylglucamine, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM iodoacetamide, 20 µg ml⁻¹ leupeptin, and Roche cOmplete Mini Protease cocktail in PBS) and rocked for 1 h at 4°C. The cell lysate was spun in a centrifuge at 20,000 g for 30 min at 4°C. To eliminate nonspecific binding of peptides, the supernatant was first incubated with polyclonal mouse IgG (1.5 mg antibody per sample; Bio X Cell) bound to sepharose 4B at 4°C for 30 min. The unbound material containing MHC-II–peptide complexes was collected and was then added to a tube containing PBS-washed sepharose conjugated to the anti–I-A^g7 antibody (1.5 mg per sample; AG2.42.7) and incubated at 4°C overnight. The I-A^g7–sepharose was applied to a column and washed four times as follows: 10 ml 150 mM NaCl and 20 mM Tris, pH 7.4; 10 ml 400 mM NaCl and 20 mM Tris, pH 7.4; 10 ml 150 mM NaCl and 20 mM Tris, pH 7.4; and 10 ml 20 mM Tris, pH 8.0. Peptides were eluted with 10% acetic acid and dried with a SpeedVac. Eluted peptides were passed over detergent removal spin columns (Pierce) to remove traces of remaining detergent and were cleaned using C18 Spin Columns from Thermo Fisher Scientific (Pierce).

In‐solution digestion was performed before MS analysis. Twenty μg proteins were diluted to a final volume of 20 μL with digestion buffer (50 mM NH₄HCO₃, pH 8.0). The sample was treated with 1 μL of 1 M DTT, mixed well, and incubated at 50°C for 15 min, followed by the addition of 1 μL of 550 mM iodoacetamide for alkylation in darkness for 15 min at 25°C. Subsequently, 80 μL of digestion buffer and 1 μg of trypsin (Promega, Madison, USA) were added to the sample followed by incubation overnight at 37°C. The reaction was terminated by the addition of 33 μL of buffer (2% trifluoroacetic acid, 20% acetonitrile). Finally, the sample was purified by using C18 spin columns (Thermo Fisher Scientific, Rockford, USA) according to the manufacturer's instructions and dried with a vacuum centrifuge (Thermo Fisher Scientific, Asheville, USA).

Samples were prepared for mass spectrometry by using WGA enrichment of GlcNAc-peptides. The WGA agarose beads (75 μL, Vector Laboratories, in Spin column-Screw caps, Pierce) were washed three times with wash buffer (100 mM Tris/HCl, pH 7.7) and spin down (12,000 rpm, MiniSpin Eppendorf), 5 min at 4°C. Total proteins (200 μg of total RM extract) were resuspended in 300 μL of wash buffer and incubated with WGA beads 2 h, at 4°C and 450 rpm (Thermomixer, Eppendorf). After 2 washes with wash buffer, GlcNAc-enriched proteins were eluted in Laemmli buffer, boiled and resolved on 1-D electrophoresis as described above. Bands were cut out and treated for trypsin digestion as described in Guiraudie et al. (19 (link)). Resulting peptides were desalted in reverse-phase C18 Spin columns (Thermofisher) and dried in Speed-Vac until use.