Biology Reference
In-Depth Information
1. Wash gel pieces with 500
L of wash solution and incubate at
RT for 15 min with gentle agitation.
2. Remove solution with a pipette carefully.
3. Wash gel pieces two more times with 500
μ
3.4 In Gel Protease
Digestion
L of wash solution
for 15 min or until the Coomassie dye has been removed.
4. Dehydrate the gel pieces in 100 % acetonitrile for 5 min. Note:
When dehydrated, the gel pieces will have an opaque white
color and will be signifi cantly smaller in size.
5. Remove acetonitrile with pipette and dry at RT for 5-10 min.
6. While gel pieces are drying prepare protease digestion
solution.
7. Rehydrate the gel with a minimal volume of protease digestion
solution (
see
Note 7
).
8. Spin down sample by centrifugation.
9. Add 25-50
μ
L of extraction solution to gel pieces and agitate
gently by vortexing at lowest setting for 10 min.
10. Spin down sample by brief centrifugation (12 kg for 30 s).
11. Transfer the supernatant (containing tryptic peptides) to the
tube.
12. Re-extract the gel plugs with an additional 25-50
μ
μ
L of extrac-
tion solution.
13. Spin down sample and transfer supernatant to tube from
step 11
.
14. Dry the pooled extracted peptides by centrifugal evaporation
to near dryness. Do not use heat. Do not dry for extended
time.
15. Store in −80 °C until analyzed by mass spectrometry for pro-
tein identifi cation.
4
Mass Spectrometry Strategy and Results Obtained
The approach used for the identifi cation of proteins presented in
excised gel pieces was based on tandem mass spectrometry (MS/
MS). Briefl y, gel pieces were “in-gel” digested with trypsin and sep-
arated using nanoAcquity UPLC system (Waters, Milford, MA,
USA). This system contained reverse-phase chromatography using
a BEH 130 C18 column (100
m particle size;
Waters, Milford, MA, USA). Separated tryptic peptides were ion-
ized by nanospray and analyzed by Q-TOF Premier MS/MS instru-
ment (Waters, Milford, MA, USA). For data acquisition, the MS
E
method was used that is based on alternate scans at low and high
collision energies. The MS data were searched against plant UniProt
databases using ProteinLynx Global Server v. 2.4 (Waters, UK).
μ
m × 150 mm, 1.7
μ
