SOP Title: Soluble Sample
Preparation from BSL 2
Prokaryotic and Viral
Organisms
Prepared by:
SOP No.
BSL2_Soluble_Prep_Rev_01
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
biological safety level (BSL) 2 bacterial or viral organisms. The peptide samples
will be used for mass spectrometry based proteomic analysis.
Responsibility
All personnel running 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 when possible low-retention microcentrifuge tubes or cryovials
are used throughout the procedure to reduce polymer leaching effects.
Ensure that samples stay on ice or in a cooling block during the procedure
unless otherwise noted.
Ensure that 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 colder
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)
100 mM NH4HCO3, pH 8 buffer
BCA or Coomassie Protein Assay Reagents
Urea (in solid form)
Thiourea (in solid form)
50 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.
Transfer supernatant from this spin to appropriate tube for soluble protein
study. (the pellet is used for the insoluble protein preparations – see
appropriate SOP). 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. Transfer the supernatant from the second spin into
the vial containing the soluble protein from the first spin.
8) Perform Coomassie or BCA Protein Assay on lysate suspension. Optimal
sample concentration is <5 mg/mL.
9) Determine volume of sample with pipet and calculate mass of protein present
in the sample.
10) Add mini Proteome as per mini Proteome calculator.
11) Add powdered form of Urea to sample to a target concentration of 7 M (424
mg/mL solution).
12) Add powdered form of Thiourea to sample to a target concentration of 2 M
(152 mg/mL solution).
13) Prepare a 50 mM solution of Dithiothreitol (DTT – 7.7 mg in 1 mL nanopure
water). Add an appropriate volume of DTT solution to obtain a 5 mM
concentration in the sample.
14) Incubate the sample at 60C for 30 min.
15) 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.
16) Dilute the sample 10-fold with 100 mM NH4HCO3 to reduce the salt
concentration.
17) Add sufficient amount of a 1M solution of CaCl2 to obtain a sample
concentration of 1 mM CaCl2.
18) Digest sample for 3 hours with Trypsin @ 37C at a concentration of 1 unit
trypsin/50 units protein.
19) 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 removed from the BSL2 hood).
20) Perform C18 SPE clean-up following the appropriate standard protocol.