StageTip-Based HAMMOC, an Effi cient and Inexpensive Phosphopeptide Enrichment Method for Plant Shotgun Phosphoproteomics - Plant Proteomics: Methods and Protocols - page 574

6. 20 % Phosphate: Dilute ortho -phosphoric acid in water to 20 %.
7. 5 % Ammonia: Dilute ammonia solution in water to 5 %.
8. 5 % Piperidine: Dilute piperidine in water to 5 %.

1. C18 200 l StageTip: packed with a single C18 disk made with

the 16 gauge needle. Can be kept at room temperature.
2. Micro-centrifuge tube.
3. The spin adaptor for micro-centrifuge tube.
4. Solution A: 80 % ACN and 0.1 % TFA in water.

μ 2.6 Desalt the

Phosphopeptides
with C18 200 l

StageTip Column

μ

5. Solution B: 5 % ACN and 0.1 % TFA in water.

6. SpeedVac.

3 Methods

3.1 Protein
Extraction and
Digestion ( See Note 3 )

1. Grind frozen plant material to a fi ne powder using a mortar
and pestle, or apparatus such as bead mills ( see Note 4 ). Make
sure that the samples are constantly frozen after harvesting
until suspended in the extraction buffer in the next step.
Proteins can be rapidly dephosphorylated upon cell disruption
without denaturants or phosphatase inhibitors.
2. Add denaturing extraction buffer to the powder and mix thor-
oughly ( see Note 1 ). Make sure that all the powder comes in
contact with the extraction buffer before the powder is thawed.
Transfer the sample to a centrifuge tube if necessary. Do not
heat the sample over 37 °C to avoid unwanted artifi cial protein
modifi cations by a urea derivative.
3. Spin down cell debris and collect supernatant.
4. Determine protein concentration of the extract.
5. Add reduction buffer to the extract (1 g

protein ( see Note 5 )) and incubate for 30 min at room tem-
perature (25 °C).
6. Add alkylation buffer to the reduced sample (1

μ l buffer for every 50 μ

μ l buffer for

g protein ( see Note 5 )) and incubate in the dark for
20 min at room temperature (25 °C).
7. Add LysC to the alkylated sample (1

every 50 μ

g protein
( see Note 6 )) and incubate for 3 h to overnight ( see Note 7 ) at
room temperature (25 °C).
8. Dilute the LysC digest with 4 volumes 50 mM NH 4 HCO 3
( see Note 8 ).
9. Add Trypsin to the diluted sample (1

μ g for every 50 μ

g pro-
tein) and incubate for overnight at room temperature (25 °C).

μ g for every 50 μ