Bacterial strain proteome sample preparation

Reagent preparation for protein extraction

using HCl adjust pH to 8, 450μl lysis buffer + 50μl 1M DTT when using

Protein extraction

Day 1

1. Cells were harvested by centrifugation for 1 min at 4℃ and 10000 × g in a 1 mL tube.

2. The supernatant was discarded, and the pellet was washed twice with 1 mL pH 7.0 10 mM Tris-HCl buffer, the following centrifugation at 4℃ at 10000 g. Cells were suspended in 0.5 mL lysis buffer (450 μl lysis buffer + 50 μl 1M DTT when using, containing 100 mM Tris-HCl, 4% SDS, 0.1 M DTT), and sonicated on ice for the 30s with pulse for 5 times. The 0.5 mL supernatant was collected by centrifugation for 1 min at 4℃ and 10000 × g.

3. 0.5 mL chilled 50% trichloroacetic acid (TCA) was added to achieve 25% TCA as the final concentration. Tubes were inverted gently and kept at -10℃ overnight to precipitate proteins present in the solution.

Day 2

1. Samples were centrifuged at 20800 × g for 20 min to obtain a concentrated protein pellet and the supernatant was discarded.

2. Protein pellets were washed with 1 mL chilled (-10℃) 80% acetone with 1 mM PMSF and 0.07% mercaptoethanol followed by centrifugation at 20800 × g for 10 min. 

3. Protein pellets were gently disaggregated in acetone by vortexing during the first acetone addition. After centrifμgation, acetone was gently removed and discarded, and a fresh 1 mL of 80% acetone with 1 mM PMSF and 0.07% mercaptoethanol was added. Then protein pellets were washed with 1 mL chilled (-10℃) 100% acetone followed by centrifugation at 20800 × g for 10 min to get precipitate.

4. Protein pellets were freeze-dried. 

Dissolve protein

1. Use a maximum of 1 mL 8M urea-Tris-HCl solution to resolubilize protein pellets. Add DTT solution to reach 5 mM (585 μL urea-Tris-HCl solution add 3 μL 1M DDT solution). Vortex 20 min to dissolve protein. Remove bubble by centrifugation for 10 min at 10000 g

2. Add iodoacetamide solution to reach 20 mM (585 μL urea-Tris-HCl solution add 12 μL 1M iodoacetamide solution). Vortex for 10 S. Incubates in dark at RT for 30 min. Centrifμge for 5 min at 10000 × g and divide supernatant into three 1 mL tubes (200 μL for each).

3. Use BCA assay to measure the protein concentration.

BCA protocol

protein dilution concentration

Mix 5 mL solution A with 0.1 mL solution B (ratio = 50:1) to make solution WR (enough for 20 samples).

Add 10 µL of each unknown sample and standard sample to plate (sample to WR ratio = 1:20).

Add 200 µL of the WR to each well and mix the plate thoroughly on a plate shaker for 15 seconds.

Incubate at 37°C for 30 minutes.

Measure the absorbance at or near 562 nm on a plate reader.

Reagent preparation for Digest

Day 3

Digest protein

1. Add 200 µL of 8 M urea in 50 mM ABC (ammonium bicarbonate) solution to the filter unit of 30 kDa filter tube. Add 50 μg protein to the filter unit. Add 100 µL of 8 M urea in 50 mM ABC solution to the filter unit and mix in a shaker at the lowest speed for 30 S. Centrifuge at 14000 × g at 25℃ for 15 min and discard the flow-through in the collection tube.

2. Add 100 µL of 50 mM IAM in 50 mM ABC solution and mix in a shaker for 1 min and incubate for 30 min in dark. Centrifuge the filter units at 14000 × g at 25℃ for 10 min and discard the flow-through in the collection tube.

3. Add 300 µL of 50 mM ABC solution to the filter unit and centrifuge at 14000 × g f at 25℃ or 10 min. Repeat this step twice.

4. Transfer the filter units to new collection tubes. Add 100 µL 50 mM ABC solution to the filter unit. Add 2 μL trypsin solution (0.5 mg/mL ) to the filter unit (enzyme to protein ratio 1:50) and mix in a shaker for 1 min. Incubate the tubes at 37℃ for 4 h.

5. Add 2 μL trypsin solution (0.5 mg/mL ) to the filter unit (enzyme to protein ratio 1:50) and mix in a shaker for 1 min. Incubate the tubes at 37℃ for 8 h.

6. Centrifuge at 14000 × g for 10 min. Add 30 µL of 50 mM ABC solution to the filter unit and centrifuge at 14000 × g for 10 min. Repeat once and discard filter units. If the peptide is dried, add 50 µL of 50 mM ABC solution and vortex. Centrifuge at 14000 × g for 10 min. Repeat until 150 μL solution is obtained.

7. Freeze at -80℃ or add 6.3 μL (0.042x) 10% TFA to about 150 μL peptide solution in the collection tube to end digest.

Reagent preparation for Desalt

Day 4

Desalt

A. Sample Preparation

Each Pierce C18 Spin Column can process 10-150 µL of a sample. Mix 3 parts sample to 1 part Sample Buffer. The final sample will contain 0.5% TFA in 5% ACN (add 50 µL sample buffer to 150 µL sample).

**B. Column Preparation **

1. Tap column to settle resin. Remove top and bottom cap. Place column into a receiver tube.

2. Add 200µL of Activation Solution to rinse walls of the spin column and to wet resin.

3. Centrifuge at 1500 × g for 1 minute. Discard flow-through.

4. Repeat steps B.2-B.3.

5. Add 200µL Equilibration Solution. Centrifuge at 1500 × g for 1 minute. Discard flow-through.

6. Repeat step B.5.

**C. Sample Binding **

1. Load sample on top of the resin bed.

2. Place the column into a receiver tube. Centrifuge at 1500 × g for 1 minute.

3. To ensure complete binding, recover flow-through and repeat steps C.1-C.2 twice.

Note: Flow-through may be retained to confirm sample binding.

**D. Wash **

1. Place the column into a receiver tube. Add 200µL Wash Solution to column and centrifuge at 1500 × g for 1 minute. Discard flow-through.

2. Repeat step D.1.

**E. Elution **

Place column in a new receiver tube. Add 20µL of Elution Buffer to the top of the resin bed. Centrifuge at 1500 × g for 1 minute and repeat step once with same receiver tube. Gently dry the sample in a vacuum evaporator.