In-Gel Tryptic Digestion and HCD/EThcD LC-MS/MS for Mapping Autophosphorylation Sites

Materials and reagents

Biological materials

1. NEK1 knockout U-2 OS Flp-In T-REx cells (generated by CRISPR-Cas9 nickase, biallelic frameshift confirmed by MiSeq; available upon request from A.R.M.)

2. GFP-NEK1 wild-type and GFP-NEK1 kinase-dead (D146A) expression constructs (available upon request from A.R.M.)

Note: The NEK1 knockout cell line and GFP-NEK1 expression constructs are specific to the experimental system used to validate this protocol. The downstream workflow can be adapted to other kinases using an appropriate cellular background, expression system, and kinase-dead control.

Reagents

1. Colloidal Blue Staining kit (protein gel stain) (Thermo Fisher Scientific, catalog number: LC6025)

2. 1 M triethylammonium bicarbonate (TEAB) buffer (Fisher Scientific, catalog number: 15215753)

3. Bond-Breaker^TM TCEP solution, neutral pH (0.5 M) (reducing agent) (Thermo Fisher Scientific, catalog number: 77720/Fisher UK 10657344)

4. Iodoacetamide (IAA) (alkylating agent) (Sigma-Aldrich, catalog number: I1149)

5. LC-MS-grade water (solvent) (Fisher Scientific, catalog number: 10434902)

6. 100% acetonitrile, LC-MS grade (solvent) (Fisher Scientific, catalog number: 10055454)

7. Ammonium bicarbonate (compound) (Sigma-Aldrich, catalog number: A6141)

8. Trypsin/Lys-C protease mix (enzyme) (Thermo Fisher Scientific, catalog number: A41007)

9. CaCl₂ (enzyme stabilizer) (Sigma-Aldrich, catalog number: C1016)

10. Formic acid (LC-MS solvent modifier) (Merck, catalog number: 1.00263)

11. n-Dodecyl-β-D-maltoside (DDM) (LC-MS additive/detergent) (Thermo Fisher Scientific, catalog number: 89902)

12. Trifluoroacetic acid (TFA) (LC-MS grade) (LC-MS solvent modifier) (Thermo Fisher Scientific, catalog number: 85183)

13. Pierce HeLa protein digest standard (Thermo Fisher Scientific, catalog number: 88329)

Solutions

1. 50 mM TEAB (see Recipes)

2. 120 mM TCEP (see Recipes)

3. 0.5 M iodoacetamide (see Recipes)

4. Trypsin/Lys-C digestion buffer (see Recipes)

5. 80% ACN/0.5% TFA (see Recipes)

6. Peptide resuspension buffer (see Recipes)

7. 50 mM ammonium bicarbonate (see Recipes)

8. 50 mM ammonium bicarbonate in 50% acetonitrile (see Recipes)

9. 0.1% TFA (see Recipes)

10. LC solvent A (see Recipes)

11. LC solvent B (see Recipes)

Recipes

1. 50 mM TEAB

a. To prepare 10 mL, combine 0.5 mL of 1 M TEAB stock with 9.5 mL of LC-MS-grade water.

b. Prepare fresh on the day of use.

2. 120 mM TCEP (in 50 mM TEAB)

a. Use 0.5 M Bond-Breaker^TM TCEP solution, neutral pH, as the stock solution.

b. To prepare 1 mL, combine 240 μL of 0.5 M TCEP stock with 760 μL of 50 mM TEAB.

3. 0.5 M iodoacetamide (in 50 mM TEAB)

a. To prepare 1 mL, dissolve 92.5 mg of IAA in 50 mM TEAB and adjust to a final volume of 1 mL.

b. Prepare fresh immediately before use and protect from light throughout preparation and use.

4. Trypsin/Lys-C digestion buffer

a. Prepare 50 mM TEAB containing 2.5 mM CaCl₂.

b. Resuspend trypsin/Lys-C protease mix according to the manufacturer's instructions and prepare sufficient digestion solution to provide 2 μg of enzyme per sample.

c. For each sample, prepare 100 μL of digestion solution containing 2 μg of trypsin/Lys-C.

d. Prepare fresh immediately before use.

5. 80% ACN/0.5% TFA

a. To prepare 10 mL, combine 8 mL of LC-MS-grade acetonitrile, 50 μL of TFA, and LC-MS-grade water to a final volume of 10 mL.

b. Prepare fresh on the day of use.

6. Peptide resuspension buffer

a. Prepare fresh using LC-MS-grade water and 0.1% formic acid.

b. Add n-dodecyl-β-D-maltoside (DDM) to a final concentration of 0.015% (w/v).

c. Reconstitute dried peptide pellets with 10 min of gentle shaking before transferring to a suitable vial and loading the LC autosampler.

7. 50 mM ammonium bicarbonate

a. To prepare 10 mL, dissolve 39.5 mg of ammonium bicarbonate in LC-MS-grade water and adjust to a final volume of 10 mL.

b. Prepare fresh on the day of use.

8. 50 mM ammonium bicarbonate in 50% acetonitrile

a. To prepare 10 mL, combine 5 mL of 100 mM ammonium bicarbonate (79.1 mg in 10 mL of LC-MS-grade water) with 5 mL of LC-MS-grade acetonitrile.

b. Prepare fresh on the day of use.

9. 0.1% (v/v) TFA

To prepare 10 mL, add 10 μL of TFA to LC-MS-grade water to a final volume of 10 mL. Used only for the optional C18 clean-up in Stage 6.

10. LC solvent A: 0.1% (v/v) formic acid in LC-MS-grade water

To prepare 1 L, add 1 mL of formic acid to 999 mL of LC-MS-grade water.

11. LC solvent B: 80% (v/v) acetonitrile, 0.08% (v/v) formic acid in LC-MS-grade water

To prepare 1 L, combine 800 mL of LC-MS-grade acetonitrile, 0.8 mL of formic acid, and LC-MS-grade water to 1 L.

Laboratory supplies

1. 1.5 mL protein low-binding microcentrifuge tubes (Sarstedt, catalog number: 72.706.600)

2. 1–200 μL round gel loading tips (Starlab, catalog number: I1022-0600), for removing wash solutions from around gel pieces without aspirating them

3. Disposable sterile scalpels (Swann-Morton, catalog number: DS11)

4. Clean glass plate or disposable Petri dish as a cutting surface, rinsed with LC-MS-grade water immediately before use

5. LC-MS vials and caps (Fisherbrand^TM 11 mm Snap Ring Micro-Vial, Plastic, catalog number: 11717597; VWR Snap Caps for Snap Ring Vials ND11, catalog number: 548-0016)

6. Powder-free nitrile gloves (worn at all times)

7. Aluminium foil for protecting iodoacetamide-containing samples from light

8. Microcentrifuge tube racks

9. C18 desalting tips (e.g., Waters, catalog number: WAT0200805), required only if the optional peptide clean-up step is performed

Equipment

1. ThermoMixer (Eppendorf)

2. SpeedVac vacuum concentrator (Thermo, model: Savant SPD140DDA)

3. Scalpel and clean cutting surface

4. Dry ice

5. Orbitrap Lumos Tribrid mass spectrometer (Thermo Scientific) with EASY-nESI source

6. Dionex Ultimate 3000 nano-LC system

7. EASY-Spray^TM HPLC Column (Thermo Scientific^TM, catalog number: ES903, 2 µm, 100 Å, 75 µm × 50 cm)

8. Acclaim PepMap 100 C18 trapping column (Thermo Scientific^TM, catalog number: 164564, 150 mm × 0.1 mm × 5 µm)

9. Nano-LC fittings and consumables: nanoViper fingertight fittings and PEEK/fused-silica capillary tubing (20–50 µm i.d.) for all post-column connections; use the minimum practicable tubing length to limit extra-column dispersion

Note: Recommended maintenance: replace the trapping column every ~500 injections or sooner if carryover increases; clean the ion transfer tube monthly; run a standard peptide mixture (e.g., Pierce HeLa protein digest standard) before each sample batch to confirm chromatographic and spectral performance.

Software and datasets

1. FreeStyle software v1.5 (Thermo Fisher Scientific; available via https://thermo.flexnetoperations.com/control/thmo/login), used here for XIC extraction and integration

2. Proteome Discoverer 2.4 (Thermo Scientific), used for database searching and phosphosite localisation

3. Mascot v2.8.3 (Matrix Science; requires institutional license), used as the search engine within the associated workflow

4. AlphaMap (Voytik et al. [3]; open-source Python package)

5. Skyline v25.1 (MacCoss Lab, University of Washington), used for TIC-normalised XIC analysis in the associated NEK1 study

Note: Proteome Discoverer and Mascot are commercial products and require a paid institutional licence; Mascot additionally requires access to a local or hosted Mascot server. FreeStyle is distributed free of charge by Thermo Fisher Scientific but requires registration of a user account and reads only Thermo .RAW files. Skyline is free and open source and reads Thermo, Bruker, SCIEX, Agilent, and Waters formats. Open-source alternatives: Laboratories without Proteome Discoverer/Mascot licences can perform equivalent database searching and phosphosite localisation using FragPipe/MSFragger with PTM-Prophet, MaxQuant with the Andromeda search engine and its PTM score, or the Trans-Proteomic Pipeline (Comet with PTMProphet). For XIC generation and integration, Skyline is a fully open-source substitute for FreeStyle and is the software used for the quantification reported in the associated research article [1]. Localisation-probability thresholds should be applied on the equivalent scale reported by the chosen tool.

Procedure