Arabidopsis Proteomics: A Simple and Standardizable Workflow for Quantitative Proteome Characterization - Plant Proteomics: Methods and Protocols - page 268

3. Reducing solution: 10 mM DTT in 100 mM NH 4 HCO 3 ,
pH 8.5.
4. Alkylating solution: 54 mM iodacetamide in 100 mM
NH 4 HCO 3 , pH 8.5.
5. Extraction solution: 50 % ACN, 0.1 % trifluoroacetic acid (TFA).
6. Trypsin solution: 3 ng/μl Trypsin (Promega: Sequencing
Grade Modified Trypsin V5113), 50 mM NH 4 HCO 3 , pH 8.5,
5 % ACN.
7. SpeedVac or vacuum concentrator.
8. Spatula or needle for cutting gel pieces.
9. Thermomixer.

2.5 MS Analysis

1. 2 % ACN, 0.1 % formic acid (FA).
2. Ultrasonic bath.
3. HPLC vials.
4. NanoHPLC (Dionex UltiMate ® 3000 RSL nano system with
autosampler).
5. Trap column: Acclaim ® PepMap100 (C18, 3 μm, 100 Å).
6. Analytical column: Acclaim ® PepMapRSLC (C18, 2 μm, 100 Å).
7. Solvent A: H 2 O, 0.1 % FA; solvent B: ACN, 0.1 % FA.
8. LTQ-XL, LTQ-Orbitrap XL, LTQ Orbitrap Velos, FTICR
(Thermo Scientific; NSI).
9. Software for data interpretation (see also Methods for the dif-
ferent options).
10. Arabidopsis TAIR10 database with common contaminants
included.

3 Methods

1. Precool mortar and pestle.
2. Chill extraction buffer on ice, and freshly add protease inhibi-
tors (e.g., doubly concentrated complete Protease Inhibitor
Cocktail, EDTA-free, Roche).
3. Homogenize plant tissue to a fine powder in liquid nitrogen
using mortar and pestle. Store the samples in liquid nitrogen.
4. Resuspend and defrost the powder in extraction buffer ( see
Note 6 ).
5. Transfer samples to microcentrifuge tubes.
6. Add SDS to obtain a final concentration of 4 % (w/v), and mix
gently by inversing the tube.
7. Denature proteins at 70 °C for 10 min in a heating block.

3.1 Protein
Extraction