Biology Reference
In-Depth Information
3.3 Preparation of
Samples for Analysis
of the Viral Proteome
by Mass Spectrometry
1. Lysis and protein concentration measurement: Lyse a small
aliquot of virus by adding the reagents or aliquots of stock
solutions directly to the virus solution (
see
Subheading
2.3.2
).
Sonicate 3 × 10 s at 15 micron. Use viral lysates to determine
protein concentration in your virus batch. For later MS analysis
it is necessary to estimate how much proteins are digested.
2. Lysis and digestion: Take out a desired amount of virus (con-
centration determined in
step 1
) (normally 10
3.3.1 In-Solution
Based Approach
g will be
enough for a number of replicates in total protein analysis and
50
μ
g will be suitable per phosphopeptide enrichment) and
add reagents or aliquots of stock solutions and inhibitors
directly to the virus solution (
see
Subheading
2.3.2
). Sonicate
3 × 10 s at 15 micron. Prepare 45 mM DTT solution from
stock and add 1/10 of the sample volume. Incubate 20 min at
60 °C. Prepare 100 mM IAA solution. Add 1/10 volume of
IAA solution to the sample and incubate in the dark, at room
temperature, shaking at 400 rpm. Add 0.3× lysis volume of
10× HEPES buffer and then dilute the sample to a volume
corresponding to 4× lysis volume with Milli-Q water (Example:
If 50
μ
μ
L of lysates, the fi nal volume will be 200
μ
L after dilu-
tion. Then 200 × 0.3 = 60
L of 10× HEPES should be added
and the volume of water to be added is 200
μ
μ
L—50
μ
L (lysates
volume)—5
μ
L (DTT solution)—5
μ
L (IAA solution)—60
μ
L
L). Add trypsin in a ratio 1:50 of
trypsin-protein. Digest at 37 °C, shaking at 400 rpm, over-
night. (For other enzymes, use conditions recommended by
the supplier). Proteins are now cleaved into peptides. Acidify
the sample by adding TFA to a fi nal concentration of 1 %.
3. Sample fi ltration: Filter the sample through a 10 kDa MWCO
ultrafi ltration unit to remove uncleaved and large polypeptides
that cannot be analyzed by LC-MS and instead could clog the
system. Save the fi ltrate and check that there <10 % of loaded
sample left in the upper part.
4. Sample desalting: Use for example the Stage-Tip technology
to desalt the peptides [
33
]. Punch an Empore Disks C
18
mem-
brane twice with a G20 needle and transfer the pieces into the
edge of a 200
(10× HEPES solution) = 80
μ
L pipette tip. Use a 1.5 mL Eppendorf tube
and punch the lid so the prepared tip can fi t therein. Load
50
μ
L 90 % acetonitrile, 0.1 % TFA solution and spin down.
Use table centrifuge at a maximum speed of 1,500 ×
μ
g
. Load
50
L of 0.1 % TFA and spin down, then load the sample in
50-100
μ
L 0.1 %
TFA. Place the tip in a new Eppendorf tube and elute the pep-
tides with 50
μ
L portions and spin down. Wash with 20
μ
L of 50 % acetonitrile in 0.1 % TFA Milli-Q
water. Dry the sample down in a SpeedVac and store in −20 °C
until MS analysis.
μ