Biology Reference
In-Depth Information
2.2 Buffers
and Solutions
1. Extraction buffer: 50 mM HEPES, pH 7.8, 1 mM EDTA,
1 mM KCl, and 2 mM MgCl
2
.
2. Solubilization buffer: 50 mM HEPES, pH 7.8, 8 M urea.
3. Trypsin buffer: 10 % ACN, 50 mM ammonium bicarbonate,
2 mM CaCl
2
.
4. Solvent A: 0.1 % FA.
5. Solvent B: 0.1 % FA and 99.9 % ACN.
2.3 Equipment
and Software
1. Thermo Scientifi c LTQ Orbitrap XL mass spectrometer and
Xcalibur software.
2. ProtMAX (
http://www.univie.ac.at/mosys/software.html
)
.
A detailed protocol for ProtMAX can be found elsewhere [
7
].
3. RAW to mzXML fi le converter (MassMatrix MS Data File
Conversion, v3.9).
3
Methods
3.1 Protein
Extraction
of a Medicago Leaf
Master Sample
Medicago leaves (100 mg fresh weight) were homogenized at 4 °C
in the extraction buffer. After centrifugation (10,000 ×
g
, 20 min,
4 °C), proteins in the supernatant were precipitated overnight
using ice-cold acetone and 0.5 %
-mercaptoethanol. After cen-
trifugation for 15 min at 4,000 ×
g
at 4 °C, the supernatant was
removed and the pellet air dried for 10 min.
β
The precipitated proteins were resuspended in solubilization buf-
fer. For digestion, 50
3.2 Sample
Preparation
g
endoproteinase LysC (Roche Diagnostics Corp.) during 5 h at
30 °C. Samples were then diluted to 2 M urea using trypsin buffer
and 10
μ
g of protein were incubated with 0.5
μ
l Porosozyme
®
immobilized trypsin (Applied Biosystems,
CA, USA) were added for further digestion overnight at 37 °C.
The digests were then desalted on C-18 Selpex cartridges (Varian,
CA, USA), eluted with methanol and dried in a vacuum
concentrator.
μ
For protein identifi cation, a common DDP analysis was performed
(top5 MS/MS,
n
= 3) using a 120-min gradient from 2 % solvent A
and 98 % solvent B to 60 % B. 500 ng of protein digest were loaded
to a C18 reversed monolithic column (Chromolith
®
CapRod
150 × 0.1 mm i.d., Merk KGsA, Germany). After MS analysis the
Orbitrap raw fi les were searched against a
M. truncatula
database
(DFCI release 10) using the SEQUEST algorithm of the Thermo
Proteome Discoverer 1.3 software. In order to identify with high
statistical confi dence, only precursor ions within a mass tolerance
limit of 3 ppm were allowed. Additionally, the occurrence of at least
two distinct peptides per protein is required. The output fi le was a
multi-consensus Excel sheet with the identifi ed proteins and their
3.3 Initial
Identifi cation and
Selective Peptide
Extraction
