Biology Reference
In-Depth Information
LofN
0
O
0
-bis(trimethylsilyl)-trifluoroacetamide, incubated at RT for 10 min
and diluted with 30
10
m
L ethyl acetate. GLC/MS is performed on a Hewlett-Packard
5890 gas chromatograph equipped with a mass selective detector (HP 5972) and
HP5-MS capillary column (crosslinked 5% phenyl methyl
m
siloxane) with
L are injected at 270
C
injection temperature in the splitless mode with helium as a carrier gas at a flow rate
of 0.9 mL/min (constant flow). The following temperature program can be used:
1 min at 100
C with 10
C/min to 250
C and with 3
C/min to 310
C. Mass
spectra are obtained in scan mode with 3.27 scans/s using a scan range of
200-500 amu. Individual sterols are identified according to their retention time
and mass fragmentation pattern.
30 m
0.25 mm
0.25
m film thickness. Aliquots of 1
m
m
2.2.3.6
MS of nonpolar lipids and phospholipids
A detailed protocol for mass spectrometric analysis of nonpolar lipid and phospho-
lipid species has been described by
Grillitsch et al. (2011)
. For this analysis, lipids
extracted as described above are diluted 1:100 in acetonitrile/2-propanol (5:2; v/v),
1% ammonium acetate, 0.1% formic acid. 5
M of TG (species 51:0) and phospha-
tidylcholine (species 24:0) are added serving as internal standards. Thermo Hypersil
GOLD C18 column (100
m
1 mm, 1.9 mm) with solvent A (water with 1% ammo-
nium acetate, 0.1% formic acid) and solvent B (acetonitrile/2-propanol, 5:2; v/v;
1% ammonium acetate; 0.1% formic acid) are used for chromatographic separation
of lipid species. The gradient changes from 35% to 70% solvent B within 4 min, and
further to 100% solvent B in 16 min. These conditions are held constant for 10 min
with a flow rate of 250
L/min. MS is performed by HPLC direct coupling to a FT-
ICR-MS hybrid mass spectrometer (LTQ-FT, Thermo Scientific) equipped with an
IonMax ESI source. The mass spectrometer is operated at a mass accuracy of
<
m
2 ppm with external calibration and resolution of 200,000 full width at half
height at 400
m/z
. The spray voltage is set at 5000 V, the capillary voltage at
35 V, the tube lens at 120 V, and the capillary temperature at 250
C. Peak areas
are calculated by QuanBrowser for all lipid species identified according to their exact
mass and retention time.
2.3
RESULTS AND DISCUSSION
2.3.1
SDS-PAGE and Western blot
LD are present at low abundance in yeast cells grown under standard conditions.
Nevertheless, they can be isolated at high purity without significant contamination
by other organelles such as mitochondria or microsomes. Yeast LD contain a distinct
and characteristic set of proteins which can be used for the quality control of isolated
LD fractions. To test the enrichment of LD over the homogenate, the sterol 24-C-
methyltransferase Erg6p can be employed as a suitable marker protein. Contamina-
tion of the LD fraction with other organelles is usually low as can be seen by the
analysis of marker proteins characteristic for various organelles. A Western blot
