Biology Reference
In-Depth Information
a
0.50
0.40
0.30
0.20
0.10
0
2.10
1.00
b
Golgi
Endoplasmic reticulum
Plasma membrane
Peroxisome
1.80
0.80
1.50
0.60
1.20
0.90
0.40
0.60
0.20
0.30
0
0
0
8
16
24
32
40
48
56
64
72
FFE fraction number
Fig.
2
Distribution of endomembrane fractions after ZE-FFE and analysis by mass spectrometry. (
a
) Total
protein after ZE-FFE separation (96-well plate) measured at
A
280
nm from a sample prepared according to
outlined procedures. (
b
) The occurrence of relative abundance of organelle marker proteins considered to be
robustly representative of particular subcellular compartments in fractions after ZE-FFE. A selection of fractions
was analyzed by mass spectrometry and data interrogated against Mascot. Identifi ed proteins were quantifi ed
by spectral counting using the Scaffold 3 software. The spectral counts for proteins selected as markers for
traffi cking and destination compartments (ER, Golgi, PM) were aligned along with contaminating organelles
(peroxisome), demonstrating the extent of separation of the ER and Golgi compartments using the techniques
and parameters detailed in these methods
for assigning proteins more confi dently to compartments that are
particularly challenging to separate by coupling FFE, mass spec-
trometry, and bioinformatics techniques. Such an approach was
recently applied to proteins showing a strong pattern of co-migration
with known Golgi markers during FFE [
5
].
4
Notes
1. Media can be dispensed into 250 mL Erlenmeyer fl asks as
90 mL aliquots and sealed with foil prior to autoclaving.
2. A variable-speed benchtop orbital shaker with a large orbital
throw is optimal (at least 2 cm); this allows slow rotation while
maintaining the cells in solution. This permits effi cient enzymatic
digestion of the cell wall.
3. DTT is added immediately prior to use. A stock solution can
be stored at −20 °C.
4. All FFE buffers must be used within 24 h and can be stored at
4 °C. Triethanolamine should be weighed. A generous volume
of FFE buffer 3, e.g., 800 mL for a 1-2-h run, is recommended
