SOP Title:
SOP No.
BSL2_Global_Prep_Rev_01
Prepared by:
Heather Mottaz
Effective:
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 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 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
60 ºC incubator
37
ºC 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) 100 mM NH
4
HCO
3
, pH 8 buffer
2) BCA or Coomassie Protein Assay Reagents
3) Urea (in solid form)
4) Thiourea (in solid form)
5) 50 mM Dithiothreitol (DTT)
6) 1 M CaCl
2
7) Sequencing-grade modified Trypsin
Procedure
1) Thaw pelletized cells and reconstitute in 100 mM NH
4
HCO
3
, 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) Perform Coomassie or BCA Protein Assay on lysate suspension. Optimal sample concentration is <5 mg/mL.
7) Determine volume of sample with pipet and calculate mass of protein present in the sample.
8) Add mini Proteome as per mini Proteome calculator.
9) Add powdered form of Urea to sample to a target concentration of 7 M (424 mg/mL solution).
10) Add powdered form of Thiourea to sample to a target concentration of 2 M
(152 mg/mL solution).
11) 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.
12) Incubate the sample at 60

C for 30 min.
13) 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.
14) Dilute the sample 10-fold with 100 mM NH
4
HCO
3
to reduce the salt concentration.
15) Add sufficient amount of a 1M solution of CaCl
2
to obtain a sample concentration of 1 mM CaCl
2
.
16) Digest sample for 3 hours with Trypsin @ 37

C at a concentration of 1 unit trypsin/50 units protein.
17) After trypsin incubation, immediately place sample on ice or quick freeze in liquid nitrogen and store at -80
ºC until ready for cleanup step (at this point the sample can be removed from the BSL2 hood).
18) Perform C18 SPE clean-up following the appropriate standard protocol.