Biology Reference
In-Depth Information
2.2.2.2
Purity control by Western blot analysis
After precipitation of the desired amount of protein as described above, the resulting
pellets are suspended in SDS-loading buffer for analysis by SDS-PAGE using 12.5%
separation gels (
Laemmli, 1970
). Western blot analysis for testing the purity of iso-
lated LD is performed routinely according to the method of
Haid and Suissa (1983)
using rabbit antibodies against marker proteins representing various yeast organelles,
such as Erg1p (LD, ER), Erg6p (LD, ER), Ayr1p (LD, ER), Prc1p (vacuoles), Fox1p
(peroxisomes), Por1p (mitochondria), Cyb2p (mitochondria), and Wbp1p (ER).
Peroxidase-conjugated secondary antibody and enhanced chemiluminescent signal
detection reagents (SuperSignal
™
, Pierce Chemical Company) are used to visualize
immunoreactive bands.
2.2.2.3
MS of proteins
Different protocols for LD proteome analysis by MS have been published
(
Athenstaedt et al., 2006; Ayciriex et al., 2012; Grillitsch et al., 2011; Ivashov
et al., 2012
). A typical analysis of LD proteins from
S. cerevisiae
was described
by
Grillitsch et al. (2011)
. One hundred micrograms of proteins are precipitated
by TCA as described above, and the resulting pellets are dissolved in 100
Lof
25 mM NH
4
CO
3
. To reduce disulfide bridges 45 mM DTT is added and the solution
is incubated for 1 h at 60
C with shaking at 400 rpm. After cooling down to RT,
cysteine residues are alkylated in the presence of 100 mM iodoacetamide for
45 min in the dark. This reaction is quenched after 45 min by adding 12.5
m
L
45 mM DTT and another incubation step for 45 min. Trypsin digestion for obtaining
suitable peptides for further analysis is carried out at an enzyme to protein ratio of
1:50 (w/w) for 18 h at 37
C, which is stopped by addition of 1
m
L of 10% TFA.
m
Then, samples are concentrated in a Speedvac to approximately 8
L and diluted
m
to 15
L with solvent 1 (8% ACN and 0.1% TFA). Separation of tryptic fragments
is performed by nLC on a ProxeonBiosystems EASY-nLC
m
system coupled to a
SunCollect MALDI spotting device (SunChrom). This method is also referred to
as shotgun proteomics. Alternatively, precipitated proteins are separated by SDS-
PAGE, bands are excised, and proteins are digested with trypsin. The risk of this
method is that proteins present at low abundance may be lost during electrophoretic
separation. It has to be noted, however, that also the shotgun proteomics method has
disadvantages, such as the missing chance to detect isoforms of proteins.
For desalting, samples are loaded onto a packed 100
™
m
30 mm precolumn
m
BEH 180 C18300
˚
filled with Waters X-Bridge
™
3.5
m
m for 15 min with 30
m
L
of solvent 1. Peptides are then separated on a 100
m
m
150 mm column (Waters
BEH 180 C18300
˚
3.5
X-Bridge
m) at a flow rate of 400 nL/min. The elution
gradient is linearly increased from 8% to 45% solvent 2 (92% ACN, 0.1% TFA)
within 100 min, to 90% solvent 2 within 20 min which is held for further 10 min
before switching to 8% solvent 2 within 5 min which is held for another 5 min.
The LC-eluent is then mixed with matrix solution containing 3.5 mg/mL alpha-
cyano-4-hydroxycinnamic acid (BrukerDaltonics) dissolved in 70% ACN and 0.1%
TFA, containing 60 fmol [Glu
1
]-fibrinopeptide B (Bachem) as internal standard.
™
m
