Biology Reference
In-Depth Information
The MALDI spotting is performed every 20 s on a blank 123
81 mm Opti-TOF
™
LC/MALDI Insert metal target (ABSciex). Mass spectra are acquired on a 4800
TOF/TOF
Analyzer (ABSciex) equipped with a Nd:YAG laser, emitting at
355 nm at a frequency of 200 Hz. All spectra are obtained in the positive reflector
mode between 700 and 4500
m/z
with fixed laser intensity. 750 laser shots per spot
are accumulated. Fragmentation is conducted with 1 kV collision energy using air as
collision gas. Conditions for MS/MS must be optimized regarding sample consump-
tion and multiple selections of identical precursors. The MS/MS precursor selection
is carried out via the instrument's software with a total of 6 precursors per spot with a
minimum signal-to-noise ratio of 80 for fragmentation. Matrix signals can be
almost eliminated by excluding potential matrix clusters between 700 and 1400
m/z
(decimal values 0.030
™
0.1
m/z
). For protein and peptide identification a
Mascot Database search engine v2.2.03 (Matrix Science Ltd.) and the Swissprot Pro-
yeastgenome.org
)
can be used.
2.2.3
Lipid analysis of LD
2.2.3.1
Lipid extraction
Lipids are extracted from LD for further analysis using the method of
Folch, Lees,
and Sloane Stanley (1957)
. An aliquot of LD samples is extracted in 3 mL chloro-
form:methanol (2:1; v/v) in a Pyrex glass tube by vortexing for 1 h at RT. Proteins are
removed by adding 1.5 mL of 0.034% MgCl
2
. After incubation for 30 min, the
extract is centrifuged at 1500 rpm for 5 min at RT. An upper aqueous phase, a protein
containing interface layer, and a lower organic phase are formed. The aqueous phase
and the interface layer have to be removed. Alternatively, the lower organic phase
can be transferred into a new Pyrex glass tube by sucking off the lower layer with a
Pasteur pipette. To avoid contamination of the lipid extract with proteins the extract
has to be washed several times. First, 1.5 mL of 2 N KCl/MeOH (4:1; v/v) is added to
the organic phase and shaken on a Vibrax for 10 min. After centrifugation at
1500 rpm for 5 min, the upper phase is removed. Then, another washing step with
an artificial upper phase (chloroform/methanol/water; 3:48:47; per vol.) follows,
which can be repeated several times depending on the required purity of the extract.
After shaking on the Vibrax for 10 min and centrifugation at 1500 rpm for 5 min fol-
lowed by removal of the aqueous phase the final lipid extract is dried under a stream
of nitrogen and stored at
20
C.
2.2.3.2
Thin layer chromatographic analysis of nonpolar lipids
For the analysis of nonpolar lipids, for example TG and SE, an aliquot of LD is
extracted as described above and dissolved in chloroform/methanol (2:1; v/v). An
equivalent to 0.2-0.5
g protein is spotted onto a Silica Gel 60 TLC plate (Merck)
with a Hamilton syringe or a TLC sample applicator (CAMAG). Additionally, 1, 5,
10, and 15
m
g of triolein, ergosterol, and cholesteryl oleate are spotted onto the plates
as standards for quantification.
m
