Jan 20, 2026

Public workspaceSample preparation protocol for Mass Spectrometry Analysis

DOI
https://dx.doi.org/10.17504/protocols.io.14egn9ekql5d/v1
  • Manon Gilson1,
  • Guillaume Bayon-Vicente1,
  • Simone Krings1,
  • Ruddy Wattiez1,
  • Baptiste Leroy1
  • 1UMons
Manon Gilson
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn9ekql5d/v1
Protocol Citation: Manon Gilson, Guillaume Bayon-Vicente, Simone Krings, Ruddy Wattiez, Baptiste Leroy 2026. Sample preparation protocol for Mass Spectrometry Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9ekql5d/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: January 09, 2025
Last Modified: January 20, 2026
Protocol Integer ID: 118003
Keywords: Protein, extraction, mass spectrometry, sample preparation protocol for mass spectrometry analysis, protein samples suitable for further proteomic analysis, using mass spectrometry, mass spectrometry analysis, further proteomic analysis, protein sample, detailed workflow for protein extraction, sample preparation protocol, protein extraction, enzymatic digestion, trypsinolysi
Abstract
This protocol outlines a detailed workflow for protein extraction, quantification, reduction, alkylation, precipitation, and enzymatic digestion (trypsinolysis). This protocol will generate protein samples suitable for further proteomic analyses using mass spectrometry.
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Materials



Troubleshooting
Sample preparation protocol for Mass Spectrometry Analysis
Solutions to prepare





Protein extraction
  1. If necessary, wash cells with 1 mL of PBS 1X
  2. Centrifuge samples at 10,000 rpm for 10 minutes and discard the supernatant
  3. Add 50 µL of guanidine hydrochloride to small pellets and 100 µL to larger pellets
  4. Perform ultrasonication on the samples (pulse 1, amplitude 20%) for three cycles of 10 seconds each and place the samples on ice between each cycle
  5. Centrifuge samples at 10,000 rpm for 10 minutes
  6. Collect supernatants and store at -20°C

Protein dosage
In a 96 well plate:
Prepare the standard curve with different dilutions of the BgG in triplicates:

  1. Dilute the samples if necessary
  2. Add 2 µL of samples and 23 µL of water to each well, in triplicates
  3. Add 225 µL of diluted Bradford reagent to each well
  4. Measure the absorbance at 595 nm
  5. Plot the OD values and the standard protein concentrations
  6. Determine protein concentration of samples (multiply by the dilution factor) by using the standard curve
Reduction
  1. Collect 50 µg of proteins and adjust the volume with guanidine hydrochloride to reach the volume of the least concentrated sample
  2. Add 1 µL of Dithioerythritol for 20 µL of sample
  3. Vortex briefly and incubate for 20 minutes at 56°C and 450 rpm
Alkylation
  1. Add 1 µL of Iodoacetamide for 21 µL of sample
  2. Vortex briefly and incubate for 30 minutes at room temperature, in the dark
Protein precipitation
  1. Dilute samples 2X with MS water
  2. Add 4 times the sample volume of cold acetone to each sample
  3. Incubate for a minimum of 1 hour at -20°C (overnight is recommended for better protein precipitation)
  4. Centrifuge at 13,000 rpm for 20 minutes at 4°C
  5. Discard the supernatant and let the residual acetone evaporate for maximum 10 minutes.
Trypsinolysis
  1. Prepare a trypsin solution of 0.05 µg/µL with the NH4HCO3 buffer (50mM)
  2. Add 20 µL (1 µg) of trypsin to digest 50 µg of proteins (avoid making bubbles)
  3. Incubate overnight at 37°C at 450 rpm
  4. Stop trypsinolysis with 5 µL of a 0.5% formic acid solution
  5. Keep the samples at -80°C for long-term stotage and -20°C for short-term storage
  6. Before injection, centrifuge the samples at 13,000 rpm for 20 minutes to pellet any remaining residues