SP3-C18 sample processing for mass spectrometry based proteomics

SP3-C18 sample preparation Witold Szymanski, Johannes Graumann 2025-11-10 Table of contents 1 Abstract 1 2 Solvents: 1 2.1 Suppliers: ................................................ 2 2.2 Instrumentation ............................................. 2 3 Sample preparation: 2 1 Abstract This protocol is adapted from the original published as “Single-pot, solid-phase-enhanced sample preparation for proteomics experiments” by Hughes et al. (2019), describing a protocol using paramagnetic beads and a hydrophilic interaction mechanism to bind, wash and elute proteins in a one-tube workflow, enabling efficient, near-lossless processing of diverse protein samples (including from minute input) for downstream mass spectrometry-based proteomics. Extending the published protocol, the work flow at hand uses SP3 for protein extraction as well as detergent removal and combines it with C18 solid phase extraction for peptide desalting, adapted from “Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips” by Rappsilber et al. (2007). 2 Solvents: • Lysis buffer - 4% SDS/SLS in 50mM TEAB. The protocol is compatible with many other detergents. Lysate may be diluted using 50 mM ABC buffer. Also ccompatible with UTU (6 M Urea/2 M Thiourea) utilizing work flows. • DTT stock - 400 mM (40x in dH2O) • IAA stock - 550 mM (40x in dH2O) • Lys-C stock - 0.5 µg/µl (facultative) • Trypsin stock - 0.2 µg/µl • TFA 10% or 20% in dH2O • TEAB buffer - 50 mM in dH2O • reconstitution buffer - 0.1% FA, 0.01% DDM (in dH2O) • SP3 bead stock - 50µg/µl in dH2O • C18 Macherey-Nagel columns or 96-well plates with C18; alternatively: StageTips (Rappsilber et al., 2007) •equilibration buffer - 0.1% formic acid in MS-grade dH2O •elution buffer - 50% acetonitrile, 0.1% formic acid in MS-grade dH2O •wash buffer - 5% acetonitrile, 0.1% formic acid in MS-grade dH2O •100% acetonitrile - activation buffer 1 2.1 Suppliers: • N-Lauroylsarcosine sodium salt - SLS: L9150 (Sigma Aldrich) - https://www.sigmaaldrich.com/DE/de/product/sigma/l9150 • Triethylammonium bicarbonate buffer - TEAB: T7408 (Sigma Aldrich) - https://www.sigmaaldrich.com/DE/de/product/sigma/t7408 • DL-Dithiothreitol - DTT: D0632(Sigma Aldrich) - https://www.sigmaaldrich.com/DE/de/product/sial/d0632 • Iodoacetamide - IAA: I6125 (Sigma Aldrich) - https://www.sigmaaldrich.com/DE/de/product/sigma/i6125 • Trypsin : 37286.02 (Serva) - https://www.serva.de/enDE/ProductDetails/5763_37286_Trypsin_MS_approved_212_205.html • Trifluoroacetic Acid - TFA: 85183 (Thermo Fisher) - https://www.thermofisher.com/order/catalog/product/de/en/85183 • Formic Acid - FA: A117 (Fischer Chemical) - https://www.fishersci.de/shop/products/formic-acid-99-0-optimalc-ms-grade-fisher-chemical/10780320 • C18 WP: 730522 (Chromabond) - Chromabond C18WP spin columns (Macherey-Nagel, Part No. 730522) • SPE MULTI 96-Monoblock CHROMABOND HR-X, 96x 50 mg, 85 µm - https://www.mn-net.com/de/spemulti-96-monoblock-chromabond-hr-x-96x-50-mg-85-m-738530.050m • dH2O - Honeywell CHROMASOLV™ LC-MS Ultra 14263 - https://www.avantorsciences.com/de/en/product/11036178/water-chromasolv-lc-ms-ultra-tested-for-uhplc-ms-riedel-de-han • Acetonitrile - ACN: 2690 (Geyer Chemsolute) - https://www.thgeyer.com/lab/shop/acetonitrile-uhplc-ms-min99-97–/chemsolute-/11647225_en • Ethanol: 5054.3 (Roth) - https://www.carlroth.com/de/en/alcohols/ethanol/p/5054.3 • 96Well Plate (for collection): 442404 (Thermo Fisher) - https://www.thermofisher.com/order/catalog/product/de/en/442404 • Sera Mag Speed Beads A: 09-981-121 - https://www.fishersci.com/shop/products/sera-mag-speedbeads-carboxylmagnetic-beads-hydrophilic/09981121 • Sera Mag Speed Beads B: 09-981-123 - https://www.fishersci.com/shop/products/sera-mag-speedbeads-carboxylmagnetic-beads-hydrophobic/09981123 • Dodecyl-β-D-maltoside - DDM (Roth) - https://www.carlroth.com/de/en/detergents/dodecyl-%CE%B2-dmaltoside-%28ddm%29/p/20l4.1 • Pierce™ BCA Protein Assay Kits (Thermo Fisher) - https://www.thermofisher.com/order/catalog/product/de/en/23225 • Pierce™ Quantitative Fluorimetric Peptide Assays (Thermo Fisher) - https://www.thermofisher.com/order/catalog/product/de/en/23290 2.2 Instrumentation • bespoke magnetic rack for 500 µl, 1.5 ml and 2 ml Eppis or bespoke magnetic rack for 96 deep well plates • Eppendorf ThermoMixer® C • Eppendorf Concentrator Plus • Thermo Scientific Fresco 21 bench-top centrifuge • Ohaus Frontier™ 5000 Multi-Pro-Serie FC5718R bench-top centrifuge 3 Sample preparation: 1. Lysis and Reduction/Alkylation 1. Add 8% SLS to a final concentration of 4% SLS • For cell pellets instead: add 4% SLS in 50 mM TEAB (dependent on the pellet size, typically between 100 and 400 µl) 2 2. Incubate in ThermoMixer with vortex for 10 min at 90°C, 1200 rpm 3. For very viscous samples (likely due to nucleic acid content), use sonication or Turbonuclease treatment (add 0.5 μl and incubate for 30 min at RT) 4. Estimate protein concentration using the Thermo BCA kit if possible (for EV samples ommit this step and assume less than 10 µg of proteins in the sample) 5. For subsequent steps aim for • 40 µg protein input for complex samples or • use the entire sample for EV samples or low amounts of sorted cells 6. Bring the sample to 10 mM DTT using the 40x DTT stock (1 μl of DTT stock per 40 μl of sample) and incubate for 10 min, at 90°C, shaking at 1200 rpm 7. Cool the sample down to RT and recollect condensate by briefly spinning down 8. Bring the sample to 15 mM IAA using the 40x IAA stock (1 μl of IAA stock per 40 μl of sample) and incubate for 30 min, at RT, in the dark 2. SP3 sample protocol - proteins 1. Add SP3 bead stock (bead to protein ratio should be 10:1 - 100 µg of beads for each 10 µg of proteins) 2. Add 100% ACN in a 1:4 sample to ACN ratio (1 vol sample, 4 vol ACN) to the sample volume, vortex and incubate off-magnet for 5 min 3. Place on magnetic rack, incubate on-magnet for 5 min 4. Discard the supernatant by pipetting 5. Wash beads using 400 μl of 80% EtOH, shake thoroughly, incubate on-magnet for 5 min 6. Invert the whole magnetic rack several times (this will also shake loose beads retained in the lid) or pipette-mix, incubate on-magnet for 5 min 7. Discard the supernatant by pipetting 8. Wash beads using 400 μl of 100% ACN, shake thoroughly, incubate on-magnet for 5 min 9. Invert the whole magnetic rack several times, incubate on-magnet for 5 min 10. Discard the supernatant by pipetting 11. Wash beads using 400 μl of 100% ACN, shake thoroughly, incubate on-magnet for 5 min 12. Invert the whole magnetic rack several times, incubate on-magnet for 5 min 13. Discard the supernatant by pipetting 14. Initiate peptidolysis in 50 μl of 50 mM TEAB using 1/50 protein input of Trypsin stock 15. Incubate overnight in ThermoMixer with a custom SP3 program (5 min at 1800 rpm, 6h at 1400 rpm, 5 min at 1800 rpm, continue using 1400 rpm, all steps at 37°C) 16. Briefly spin down the sample to collect condensate from the lid 17. Place on magnetic rack, incubate on-magnet for 5 min 18. Transfer the supernatant to a new vial or 96-well plate 19. Stop the digestion by acidifying using TFA to a final concentration of 1.5% TFA (pH must be < 2) 20. Let samples rest for 10 min at room temperature 3. Desalting peptides in the supernatant using C18 columns 1. Equilibrate the C18 columns (or HR-X-96-well plates): • Ohaus Frontier™ 5000 Multi-Pro-Serie FC5718R bench-top centrifuge for 96-well plates • Ohaus Frontier™ 5000 Multi-Pro-Serie FC5718R bench-top centrifuge for C18WP columns, placed in a bespoke column holder fitting a Swing Out Rotor 2 × 3 MTP (Buckets included) suitable for deep-well plates 1. Add 500 μl 100 % acetonitrile to the column, centrifuge at 300 rpm for 1 min 2. Add 500 μl 100 % acetonitrile to the column, centrifuge at 300 rpm for 1 min (repetition is essential!) 3. Add 500 μl equilibration buffer to the column, centrifuge at 300 rpm for 1 min 4. Add 500 μl equilibration buffer to the column, centrifuge at 300 rpm for 1 min 2. Load the samples onto the column. Centrifuge at 300 rpm for 1 min (extend time of centrifugation as necessary to achieve complete loading) • in low protein amount samples (e.g. from EVs), collect the flow-through and re-load it onto the column 3 3. Wash the column with 500 μl wash buffer, centrifuge at 300 rpm for 1 min 4. Place the column into a new Eppendorf tube (or on top of a new 96-well plate) 5. Elution 1. Add 100 μl elution buffer to the column 2. Incubate for 5 min at RT 3. Centrifuge at 300 rpm for 1 min 4. Repeat the three elution steps above 5. Dry the sample in a Eppendorf Concentrator plus (SpeedVac), at highest vacuum settings with side heating to 60 degrees and max rotation 6. For samples from 40 μg protein input, resuspend samples in 80 μl of 0.1% FA, 0.01% DDM 7. Estimate peptide concentration using the Pierce Quantitative Fluorometric Peptide Assay 8. Adjust the sample peptide concentration depending on the mass spectrometer used downstream: • 50 ng/μl for Bruker TimsTof Ultra • 150 ng/μl for Bruker TimsTof Pro • 300 ng/μl for Orbitrap Instruments 9. Transfer 50 μl of the sample to a mass spectrometry vial 10. For storage, freeze samples at -20°C or keep at 4°C for short-term storage. 4