TITLE: Chloroform-Methanol Protein Extraction for Gram-negative Bacteria (High Throughput)
AUTHORS: Jennifer Gin, Yan Chen, Christopher Petzold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven  progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significantly under different experimental conditions. Consequently, it is important to have reproducible sample preparation methods that consist of mixing, various centrifugation and incubation steps, and an extended tryptic digestion step. We developed a high-throughput sample preparation workflow that consists of cell lysis, protein precipitation, protein resuspension, protein quantification, and normalization of protein concentration followed by standard bottom-up proteomic procedures of reducing and blocking cysteine residues and tryptic digestion.</div><div class = "text-block"><span>This protocol was adapted from the manual sample preparation method found in Chen, Y., et al. "Automated “Cells-To-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes."</span><span style = "font-style:italic;">Journal of proteome research </span><span>18.10 (2019): 3752-3761.</span></div></div>

[STEPS]
?. [Protein extraction]
Thaw cells at
on ice
Note: If transferring directly from active cultures, omit this step. Adapt as needed for your specific organism and culturing conditions.
?. [Protein extraction]
Transfer 2-4 OD*mLs of cells to 8-Strip PCR tubes (Axygen, Cat.#14-222-251) or a 96-well PCR plate (ThermoFisher, Cat.#AB0600).
?. [Protein extraction]
Add  of LC-MS grade Methanol (VWR Scientific, Cat.#BJLC230-2.5). Pipet to resuspend well.
80 µl
?. [Protein extraction]
Add  of Chloroform (Sigma-Aldrich, Cat.#34854). Pipet/vortex to mix.
20 µl
Use a fume hood when handling and pipetting chloroform.
?. [Protein extraction]
Add  of LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5). Pipet/vortex to mix.
60 µl
?. [Protein extraction]
Centrifuge at .
Centrifuge: 4000 33, 1 min, 25 10
?. [Protein extraction]
Carefully remove the top layer of solvent (Methanol + Water) by pipetting.
?. [Protein extraction]
Add  of Methanol.
100 µl
Tip: Break up the protein pellet by piercing it with the pipet tip, and add Methanol to the bottom of the tube or plate.
?. [Protein extraction]
Centrifuge at .
Centrifuge: 4000 33, 2 min, 25 10
?. [Protein extraction]
Carefully discard solvent (Methanol + Chloroform).
Discard in an appropriate waste container for halogenated solvents.
?. [Protein extraction]
Air-dry for .
Tip: Do not dry for longer than   or the pellet will be difficult to resuspend.
Dry samples in a fume hood.
?. [Protein extraction]
Resuspend with  .
60 µl
[Ammonium bicarbonate in 20% Methanol]
Note: Samples are typically cloudy in this step. After trypsin digestion they will be nearly clear.
?. [Protein extraction]
Store at   until ready for Protein Quantitation Assay.
-20 °C
?. [Protein Quantitation Assay (Lowry Method)]
Dilute samples 10 fold by adding to in 8-Strip PCR tubes or 96-well plate.
[Protein sample, mix well right before transfer]
[Water]
Note: The protein concentration can be determined by using several methods that are available in kits. We use the Bio-Rad DC Protein Assay (Bio-rad Laboratories, Cat.#500-0113, Cat.#500-0114) but the Bradford protein quantification assay is also commonly used. The accuracy of most protein concentration measurements can be variable, thus it is important to minimize differences in sample handling and to use replicates when quantifying the amount of protein in a sample.
?. [Protein Quantitation Assay (Lowry Method)]
Transfer 2 replicates of each of the following to Corning 96-Well Black Polystyrene Microplate (Fisher Scientific, Cat.#07-200-590):  ABCDEFGHIJKL1BlankStd 1Std 2Std 3Std 4Std 5Std 6Std 72BlankStd 1Std 2Std 3Std 4Std 5Std 6Std 731234567891011124123456789101112513141516171819206131415161718192078
[Water (Blank)]
[Pierce Bovine Serum Albumin Standard Pre-Diluted Set (Std) (ThermoFisher, Cat.#23208)]
[Diluted samples, mix well right before adding to plate (Example 1-20)]
ABCDEFGHIJKL1BlankStd 1Std 2Std 3Std 4Std 5Std 6Std 72BlankStd 1Std 2Std 3Std 4Std 5Std 6Std 731234567891011124123456789101112513141516171819206131415161718192078
?. [Trypsin Digestion (5h - 16h)]
Chemicals to prepare:• Prepare  solution by dissolving  in • Prepare  solution by dissolving   in  •Prepare   by adding  to
[Tris(2-carboxyethyl)phosphine (TCEP)]
[TCEP]
[100mM Ammonium Bicarbonate]
[Iodoacetamide (IAA)]
[Iodoacetamide]
[100mM Ammonium Bicarbonate]
[Trypsin]
[1mM HCl ]
[Trypsin]
Store TCEP, IAA, and Trypsin in -20C.IAA is light sensitive.  Store in amber tube (Fisher Scientific, Cat.#05-402-31).
?. [Trypsin Digestion (5h - 16h)]
Incubate at   for  -.
37 °C
?. [Protein Quantitation Assay (Lowry Method)]
Add  and wait   ..
[Bio-Rad DC Protein Assay Reagent A (Bio-rad Laboratories, Cat.#500-0113)]
?. [Protein Quantitation Assay (Lowry Method)]
Add   and wait   ..
[Bio-Rad DC Protein Assay Reagent B (Bio-rad Laboratories, Cat.#500-0114)]
?. [Protein Quantitation Assay (Lowry Method)]
Read plate in the microplate reader (280 nm) and calculate protein concentrations.
?. [Trypsin Digestion (5h - 16h)]
Mix protein with TCEP, IAA, and trypsin in .add add add add add up to
[Ammonium Bicarbonate (AMBIC)]
The final concentrations will be  ,  ,   , and   (1:50 trypsin:protein ratio).  Adjust as needed for your data acquisition protocols.
[protein (2.4 ug/ul)]
[TCEP (100 mM)]
[IAA (200mM)]
[Trypsin (1 mg/ml)]
[AMBIC]
[total volume]
If you do not have enough protein, you can set your final protein concentration to 1 ug/ul.add   add   add   add   add   up to
?. [Trypsin Digestion (5h - 16h)]
Store at   until ready for LC-MS/MS analysis.
-20 °C
?. [Trypsin Digestion (5h - 16h)]
Centrifuge at .
Centrifuge: 4000 33, 15 min, 4 10
?. [Trypsin Digestion (5h - 16h)]
Carefully pipet out clear liquid sample into plastic autosampler vials (ThermoFisherScientific, Cat.#03-060-016) or a  96-well plate (BIO-RAD, Cat.#HSP9601).
?. [Trypsin Digestion (5h - 16h)]
Dilute protein samples to  in
[Ammonium Bicarbonate (AB)]
Mix protein well before you dilute it.