May 28, 2026
Methionine reactivity profiling
- 1Columbia University
- Christine Chio Lab
Protocol Citation: Christine Chio 2026. Methionine reactivity profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp4ez1gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2026
Last Modified: May 28, 2026
Protocol Integer ID: 318137
Keywords: methionine reactivity profiling oxaziridine, redox state of methionine residue, methionine residue, chemical proteomic approach, based chemical proteomic approach, redox state, proteome
Abstract
Oxaziridine-based chemical proteomic approach to characterize the redox state of methionine residues across the proteome.
Materials
- 50-100 mg adipose tissues
- Dounce homogenizer
- RIPA lysis buffer
- 10 mM Tris-hydroxypropyltriazolylmethylamine
- 2 mM CuSO_4
- 30 mM sodium ascorbate
- 200 μM dialkyodiphenylsilane biotin azide
- Ice-cold methanol
- Chloroform
- 2.5% SDS/PBS buffer
- Streptavidin Sepharose High Performance beads
- 1% SDS/PBS
- 100 mM ammonium bicarbonate (ABC)
- 0.5 M TCEP
- 0.5 M iodoacetamide
- CaCl_2
- Trypsin
- 2% formic acid (FA)
- Acetonitrile:2% FA solution
- C18 column
- Buffer A: H_2O, pH 10.0
- Buffer B: 80% ACN, pH 10.0
- UltiMate 3000 (Thermo Fisher Scientific, MA, USA)
- AUR3-15075C18 column
- Spectronaut 18 (Biognosys AG, Switzerland)
- Mus musculus database
- R Statistical Software (v4.2.2, R Core Team 2022)
Sample preparation
50-100 mg adipose tissues were lysed with a Dounce homogenizer in RIPA lysis buffer. Homogenized tissues were incubated on ice for 15 min, followed by centrifugation at 12,000 × g for 15 min. The upper fat layer was removed after centrifugation. Following protein quantification, 700 μg of total protein was used for global proteome analysis or methionine labelling using 400 μM oxaziridine-alkyne (Ox3a-Alk. Supplementary Fig. 1). Samples were incubated at 25 °C for one hour with shaking at 750 rpm. To derivatize labelled methionine residues, Copper click reaction was performed with 10 mM Tris-hydroxypropyltriazolylmethylamine, 2 mM CuSO_4, 30 mM sodium ascorbate, and 200 μM dialkyodiphenylsilane biotin azide. The reaction proceeded for 1.5 h at 25 °C in the dark with agitation at 750 rpm. To remove unreacted chemicals, ice-cold methanol and chloroform were added, and the mixture was vortexed briefly. Precipitation of protein was indicated by a white, cloudy layer. Samples were centrifuged at 20,000 × g for 10 min at 4 °C, and the upper aqueous phase was carefully discarded. The protein interface was washed twice with 1,000 μL ice-cold methanol, followed by centrifugation (20,000 × g, 10 min, 4°C) and removal of the supernatant. The protein pellet was air-dried for at least 15 min. The dried protein pellet was then reconstituted in 500 μL 2.5% SDS/PBS buffer and allowed to dissolve completely. The sample was transferred to a 15 mL conical tube and diluted with PBS to reduce SDS concentration to below 0.125%. Streptavidin Sepharose High Performance beads were added, and samples were incubated overnight at 4 °C with rotation. Beads were pelleted by centrifugation at 1,700 × g for 5 min, washed sequentially (2×) with 1% SDS/PBS (1 mL), PBS (1 mL), 6 M urea (1 mL), and PBS (1 mL), with centrifugation at 1,700 × g for 3 min between washes. Beads were resuspended in 700 μL 100 mM ammonium bicarbonate (ABC). Proteins were reduced by adding 7 μL of 0.5 M TCEP and incubated at 65 °C for 15 min at 750 rpm. After cooling, 14 μL of 0.5 M iodoacetamide was added, and the mixture was incubated at 37 °C for 30 min in the dark. Next, 0.7 μL of 1 M CaCl_2 and 2 μg of MS-grade trypsin were added, and digestion proceeded overnight at 37 °C (16 h). After digestion, the beads were centrifuged again at 1,700 × g for 3 min, and the supernatant was carefully removed and transferred to fresh tubes. Beads were washed twice with PBS, and the peptides were washed with MS-grade H_2O washing. The beads were resuspended in 1 mL of 2% formic acid (FA) and incubated for 2 hours in a thermomixer (25 °C, 1250 rpm). The solution containing eluted peptides was transferred to a new 1.5 mL LoBind Eppendorf microtube after the beads were pelleted (2,000 × g, 5 min). The beads were additionally incubated with 300 μL 1:1 acetonitrile:2% FA solution for 30 min. The eluted peptide was lyophilized under speed-vac and desalted using a C18 column before mass spectrometry.
Spectrogram Library Establishment
High pH Reverse Phase Separation: The peptide mixture was re-dissolved in buffer A (buffer A: H_2O, pH 10.0, adjusted with ammonium hydroxide), and then fractionated by high pH separation using a nanoACQUITY UPLC system (Waters Corporation, MA, USA) connected to a reverse phase column (C18 column, 2.5 mm x 250 mm, 1.9 μm). High pH separation was performed using a non-linear gradient starting from 4% B to 53% B in 40 min (B: 80% ACN, pH 10.0, adjusted with ammonium hydroxide), 53% B to 68% B in 15 min, 68% B to 95% B in 5 min and maintained here for 3 min. The column was re-equilibrated at the initial condition for 17 min. The column flow rate was maintained at 2 μL/min, and the column temperature was maintained at 30 °C. The EasyPepT Frac nano automatic fraction collection system was used to collect 8 fractions, and each fraction was dried in a vacuum concentrator for the next step.
DIA Data Collection
The UltiMate 3000 (Thermo Fisher Scientific, MA, USA) liquid chromatography system was connected to the timsTOF Pro 2, an ion-mobility spectrometry quadrupole time-of-flight mass spectrometer (Bruker Daltonik, Bremen, Germany). Samples were reconstituted in 0.1% FA and 200 ng peptide was separated by AUR3-15075C18 column (15 cm length, 1.5 μm i.d, 1.7 μm particle size, 120 Å pore size) with a 60 min gradient starting at 4% buffer B (80% ACN with 0.1% FA) followed by a stepwise increase to 28% in 25 min, 44% in 10 min, 90% in 10 min and stayed there for 7 min, then equilibrates at 4% for 8 min. The column flow rate was maintained at 400 nL/min with a column temperature of 50 °C. DIA data was acquired in the diaPASEF mode. We defined 22 × 40 Th precursor isolation windows from m/z 349 to 1229. To adapt the MS1 cycle time, we set the repetitions to variable steps (2-5) in the 13-scan diaPASEF scheme in our experiment. During PASEF MSMS scanning, the collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0 = 1.6 Vs/cm^2 to 20 eV at 1/K0 = 0.6 Vs/cm^2.
DIA Database Search
Raw data were processed and analysed using Spectronaut 18 (Biognosys AG, Switzerland) with default settings. The database used was Mus musculus (version2022, 21992 entries), downloaded from Uniprot. Carbamidomethyl on cysteine was specified as the fixed modification, while oxidation on methionine was specified as the variable modification. The retention time prediction type was set to ‘dynamic iRT’. Data extraction was determined by Spectronaut based on extensive mass calibration. Spectronaut will determine the ideal extraction window dynamically depending on iRT calibration and gradient stability. Overall, (FDR) cutoff on precursor level and protein level was 1%. Decoy generation was set to ‘mutated’, which is similar to scrambled but will only apply a random number of AA position swaps (min = 2, max = length/2). The normalization strategy was set to ‘local normalization’. Peptides that passed the 1% Qvalue cutoff were used to calculate the major group quantities with the MaxLFQ method. C_14H_25N_3O_3 (m/z = 311.1957) was used as the variable modification for oxaziridine labelled methionine residues.
Differential expression analysis
The limma package (v3.54.2) was used to compare protein expression levels, while the LOESS (limma package) normalization method via Normalize^^69 was used to compare methionine reactivity, identifying differentially expressed proteins with an adjusted p value 3c 0.05. To minimize potential signal from non-adipocyte proteins, we filtered the dataset using publicly available adipocyte proteomic profiles (PXD039523)^^70^^. Following the differential expression analysis, we performed Gene Set Enrichment Analysis (GSEA) using annotated gene sets from the Molecular Signatures Database (MSigDB) to determine whether a predefined set of genes shows statistically significant, concordant differences between two biological states. FGSEA with the fgsea package (v1.28.0) was performed. We then visualized the pathway analysis results through enrichment plots, with an adjusted p value 3c 0.05 applied throughout. All analyses were performed using R Statistical Software (v4.2.2; R Core Team 2022).
Protocol references
- R Core Team 2022