Plant Mitochondrial Proteomics - Plant Proteomics: Methods and Protocols - page 486

3. HPLC-grade acetonitrile (ACN), water, trifl uoroacetic acid
(TFA), and ammonium phosphate (monobasic) (NH 4 H 2 PO 4 ).
4. Machine-specifi c MALDI plate.
5. Saturated -cyano-4-hydroxycinnamic acid (CHCA) matrix

solution. Take 150

α

L of 90 % (v/v) ACN and 0.1 % (v/v)
TFA, add a small spatula of CHCA, vortex, and then soni-
cate for 15 min. If all matrix is solubilized, then add more
CHCA and repeat sonication until undissolved matrix is vis-
ible (saturated). Centrifuge at 10,000 × g for 5 min to pellet
undissolved matrix ( see Note 13 ).

μ

6. Matrix solution (600 μ L). This contains 516 μ L 95 % (v/v)

ACN, 0.1 % (v/v) TFA, 27 μ L saturated CHCA solution,

6 μ L 10 % (v/v) TFA, and 6 μ L 100 mM NH 4 H 2 PO 4 .

B. Method using ESI-MS/MS.
1. Peptides extracted from a protein spot as outlined above .
2. HPLC-grade ACN, water, and formic acid (FA).

3. C18 column ( see Note 14 ).

1. Resuspension solution: 8 M Urea, 50 mM NH 4 HCO 3 , 5 mM
DTT, pH 8.0.
2. Vortex.
3. IAA stock solution: 100 mM Iodoacetamide.
4. Dilution buffer: 50 mM NH 4 HCO 3 , pH 8.0.
5. Digestion solution: 1

2.4.5 Gel-Free Peptide
Fractionation and
Identifi cation to Determine
Mitochondrial Purity

Sample Preparation

μ g/ μ L trypsin in 1 mM CaCl 2 .

6. Formic acid.
7. C18 spin columns (such as Pierce C18 spin columns, Thermo
Scientifi c) or C18 embedded tips (such as ZipTips, Merck
Millipore).
8. Charging and elution solution: 70 % (v/v) ACN, 0.1 % (v/v)
FA (HPLC grade).
9. Equilibration and washing solution: 2 % (v/v) ACN, 0.1 %
(v/v) FA (HPLC grade).

10. Vacuum centrifuge.

1. Peptides extracted from a protein spot as outlined in
Subheading 3.4.5 .
2. HPLC-grade ACN, water, and formic acid.
3. C18 column ( see Note 15 ).

Sample Fractionation and
Mass Spectrometry

1. Microsoft Excel is useful but not essential as is statistical pack-
age such as Analyze-it ( www.analyze-it.com ) add-on for
Microsoft Excel.

Data Analysis