SOP No.
Sample Preparation from BSL 2 BSL2_Insoluble_Prep_Rev_01
SOP Title: Insoluble Protein
Prokaryotic and Viral
Organisms
Prepared by:
Effective:
Heather Mottaz
Technical Approval by:
Supervisory Approval by:
Replaces SOP No.
NA
Retired:
Purpose
This procedure is to be used for preparation of peptide samples from
insoluble proteins from biological safety level (BSL) 2 bacterial or viral organisms.
The peptide samples will be used for mass spectrometry based proteomic
analysis.
Responsibility
All personnel performing this experiment will be responsible for reading,
understanding and complying with the provisions outlined within this method.
It is the responsibility of the appropriate manager to ensure that this
document is reviewed and updated as needed.
Safety
Before beginning any of the procedures involved in this method, each
individual must read and sign the Chemical Hygiene Plan developed for PNNL.
Material Safety Data Sheets (MSDS) for the chemicals and reagents are
available online.
All staff must have been formally trained (one-on-one) in the use of the
BSL2 hood prior to any work being performed.
Interferences
Ensure that low-retention microcentrifuge tubes or cryovials are used
throughout the procedure to reduce polymer leaching effects.
Ensure that samples stay on ice during the procedure unless otherwise
noted.
Ensure that the original sample is free from detergents. If the sample
contains a small amount of media salts or other chemicals, these may interfere
with the protein assay. Consult the instruction manual for the protein assay to
ensure that this will not occur.
Caution
Ensure that all training based in the Integrated Operations System (IOPS)
has been completed and all safety procedures are being followed.
Follow all BSL2 procedures when working with the organisms and their
proteins.
Ensure that the following steps are performed in the BSL2 hood (unless
otherwise directed) until tryptic digestion is complete. Samples can be removed
from the hood for certain steps (i.e. incubation) in sealed containers or tubes,
following appropriate decontamination steps.
Apparatus
Items needed for sample preparation are:
Vortexer mixer
0.1 mM Zirconia/Silica Beads
Low-retention microcentrifuge tubes (0.6, 1.5 or 2.0 mL)
Cryovials with externally threaded lids containing O-rings
60ºC incubator
37ºC incubator
Cooling block at 4ºC or cooler.
Reagents
Prepare all reagents in appropriate containers, preserving sterility when
necessary. Ensure that the water used to prepare solutions is of Nanopure or
Milli-Q quality (~18 megohm·cm or better).
1)
2)
3)
4)
5)
6)
7)
8)
100 mM NH4HCO3, pH 8 buffer
50 mM NH4HCO3, pH 7.8 buffer
BCA or Coomassie Protein Assay Reagents
Urea
Thiourea
200 mM Dithiothreitol (DTT)
1 M CaCl2
Sequencing-grade modified Trypsin
Procedure
1) Thaw pelletized cells and reconstitute in 100 mM NH4HCO3, pH 8 buffer
(wash cells once if necessary with buffer).
2) Transfer reconstituted cells to 2.0 mL cryovials (ensure there is no more than
~600 L in each tube). Minimize bubble formation if possible, and keep as
much of the suspension in the bottom of the tube as possible.
3) Add 0.1 mM Zirconia/Silica Beads so that there is a ~50% volume of beads in
the tube (50% beads, 50% suspension)
4) Bead beat by vortexing vigorously (max speed) for 30 seconds, then
immediately placing in cooling block for 1 minute. Repeat this step 6 times,
for a total of 3 minutes of bead beating. After the final vortexing step, allow
tubes to remain in cooling block for 5 minutes to cool thoroughly.
5) Transfer supernatant from beads (trying to not capture beads in tip) to
separate cryovial for collection (use one with an O-ring). To original vial, add
a volume of fresh buffer to completely cover beads, vortex briefly, place in
cooling block for at least 1 minute, and transfer supernatant into collection
cryovial. Repeat wash of beads until you cannot see any cloudiness in the
wash.
6) Centrifuge lysate at 4,000 rpm at 4ºC for 2 minutes to spin out any whole
cells.
7) Collect supernatant and Ultracentrifuge at 100,000 rpm for 10 minutes @ 4ºC.
Draw off supernatant from this spin and either discard or retain for soluble
protein study (see appropriate SOP). The pellet is used for the insoluble
protein preparations. I If desired, the membrane pellet can be resuspended
(using either vortexing and pipetting or sonication) in either water or buffer
and centrifuged as above again to “wash” pellet. If sonication is chosen to
resuspend the pellet, place the parafilm-covered ultracentrifuge tube inside an
o-ring cryovial to sonicate. If retained, transfer the supernatant from the
second spin into the vial containing the soluble protein from the first spin.
8) Resuspend pellet (using either vortexing and pipetting or sonication) in 100
µL of either water or buffer and perform BCA protein assay to determine
approximate protein concentration. Determine volume of sample with pipet.
9) Centrifuge pellet again at 100,000 rpm for 5 min @ 4ºC and remove
supernatant.
10) Add mini Proteome as per mini Proteome calculator.
11) Solubilize membrane pellet into 100 to 200 µL of solubilization solution (7M
urea, 2M thiourea, 1% CHAPS in 50 mM Ammonium bicarbonate, pH 7.8)
12) Prepare a 200 mM solution of Dithiothreitol (DTT – 30 mg in 1 mL nanopure
water). Add 5 µL of DTT solution for every 100 µL of sample volume to obtain
a 10 mM concentration in the sample.
13) Incubate the sample at 60C for 30 min.
14) After removing the sample from the incubator, add buffer to the trypsin vial(s)
to obtain desired concentration (i.e. add 20 uL to vial to obtain 1 ug/uL trypsin
solution), and pre-activate trypsin by incubating vial(s) for 5-10 minutes at
37ºC.
15) Dilute the sample 10-fold with 50 mM NH4HCO3, pH 7.8 to reduce the salt
concentration.
16) Add sufficient amount of a 1M solution of CaCl2 to obtain a sample
concentration of 1 mM CaCl2.
17) Digest sample for 3 hours with Trypsin @ 37C at a concentration of 1 unit
trypsin/50 units protein.
18) After trypsin incubation, immediately place sample on ice or quick freeze in
liquid nitrogen and store at -80ºC until ready for cleanup step (at this point the
sample can be worked with outside the BSL2 hood).
19) Perform SCX SPE clean-up following the appropriate standard protocol.