May 23, 2025
Sample Preparation of Hevea brasiliensis for Proteomic Analysis by LC-MS/MS
- NyokSean Lau1,
- Emiko Okubo-Kurihara2,
- Yuko Makita Nakai3,
- Minami Matsui4
- 1Universiti Sains Malaysia;
- 2Keio University;
- 3Mebashi Institute of Technology;
- 4RIKEN Center for Sustainable Resource Science
Protocol Citation: NyokSean Lau, Emiko Okubo-Kurihara, Yuko Makita Nakai, Minami Matsui 2025. Sample Preparation of Hevea brasiliensis for Proteomic Analysis by LC-MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1mwr7vr2/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 23, 2025
Last Modified: May 23, 2025
Protocol Integer ID: 218845
Keywords: preparation of hevea brasiliensis sample, proteomic analysis by lc, quantitative proteomic analysis, proteomic analysis, hevea brasiliensis sample, reproducible sample processing of complex plant tissue, tissue sample, hevea brasiliensi, sample preparation, enhanced sample preparation, sp3 method, paramagnetic sp3 bead, protein
Abstract
This protocol describes the preparation of Hevea brasiliensis samples for quantitative proteomic analysis using the SP3 method (single-pot, solid-phase-enhanced sample preparation). The tissue samples are homogenised in SDS-containing lysis buffer, then sonicated, rotated and the proteins quantified. The proteins are reduced, alkylated and bound to paramagnetic SP3 beads and then enzymatically digested with trypsin/Lys-C. After peptide salting with SDB-based reversed-phase tips, the samples are dried, reconstituted and quantified prior to LC-MS/MS analysis. This workflow enables efficient and reproducible sample processing of complex plant tissues with minimal sample loss.
Troubleshooting
Protocol Steps
Cut the sample into small pieces using sterile scissors. Add lysis buffer containing 100 mM Tris-HCl (pH 8.0), 4% sodium dodecyl sulfate (SDS), and 20 mM NaCl.
Sonicate the sample using a sealed ultrasonic homogenizer for 30 minutes, then rotate at room temperature for 24 hours.
Repeat the sonication for 30 minutes and rotate again for 24 hours.
Sonicate once more for 30 minutes, then filter the lysate using a 0.22 µm membrane filter.
Measure protein concentration using the bicinchoninic acid (BCA) assay. Adjust the protein concentration to 0.15 µg/µL with the same lysis buffer (100 mM Tris, 4% SDS, and 20 mM NaCl).
To reduce disulfide bonds, add tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 20 mM and incubate at 80°C for 10 minutes.
To alkylate cysteine residues, add iodoacetamide (IAA) to a final concentration of 30 mM and incubate in the dark at room temperature for 30 minutes.
Prepare single-pot solid-phase-enhanced sample preparation (SP3) beads by mixing equal volumes of hydrophilic and hydrophobic Sera-Mag SpeedBeads (1:1 v/v), washing them three times with distilled water, and suspending them at 8 µg/µL in distilled water.
Add 20 µL of SP3 beads to the alkylated sample, then add three volumes of ethanol. Mix at room temperature for 20 minutes.
Wash the beads twice with 80% ethanol. Add 100 µL of 50 mM Tris-HCl (pH 8.0) and mix.
Digest the proteins by adding 200 ng of Trypsin/Lys-C Mix (Promega) and incubate overnight at 37°C.
Add 20 µL of 5% trifluoroacetic acid (TFA) and sonicate the sample.
Desalt the peptides using GL-Tip SDB (GL Sciences), then dry the eluate using a centrifugal evaporator.
Dissolve the dried peptides in 2% acetonitrile (ACN) and 0.1% TFA. Sonicate the solution to aid dissolution.
Measure peptide concentration with a BCA assay. Adjust the concentration to 200 ng/µL with 2% ACN and 0.1% TFA.
Proceed to liquid chromatography–mass spectrometry (LC-MS) analysis.