TITLE: Cell lysis, detergent-free
AUTHORS: Teesha Luehr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Detergents are generally not compatible with mass spectrometers, so this is a detergent-free method of cell lysis that is compatible with mass spectrometry. Since this protocol does not have a precipitation step, it saves time and minimizes sample loss as well.</div></div>

[STEPS]
?. [Growing Cells]
Culture HeLa cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 20mM glutamine, and 1% PenStrep.
?. [Cell Lysis]
Resuspend the cell pellet(s) in
[ice cold Milli-Q water]
Perform the lysis and digestion in the   . The lysis volumes, sonification, and digestion require the larger tube volume.
?. [Reduction & Alkylation]
Add for a final concentration of
[2M tris]
?. [LysC Digestion]
Dilute the sample to a total of
[50mM NaHCO3]
This is performed to dilute the TFE.
?. [Pellet Cells]
Using a pellet cells for
[conical tube]
[at 300 g]
Pellet in a tube a minimum size of   as this tube will be used through till the end of digestion.
?. [Pellet Cells]
Wash cells with
[cold PBS]
?. [Pellet Cells]
Pellet cells for
[at 300 g]
?. [Pellet Cells]
Carefully discard supernatant
?. [Growing Cells]
Original protocol uses 5x107 cells per sample. Use 2x107 cells. Use one 15cm Petri dish or 2-3 10 cm dishes. Each 10cm dish gives 8.8x106 cells.
?. [Pellet Cells]
Store cell pellet on ice
If not performing cell lysis immediately, the pellet can be stored at
?. [Preparing Solutions and Materials Needed]
Ice Bucket
?. [Preparing Solutions and Materials Needed]
Add andup to a total of
[tris at pH 8.5]
[tris-base]
[concentrated HCl (37%)]
[in Milli-Q water]
Calculations done with https://www.cytographica.com/lab/HHTris.html.
Tris-base is in NCE 438 chemical room on the top right doubles shelf
A stock conical tube of   is stored on the bench.
?. [Preparing Solutions and Materials Needed]
Add to
[TCEP]
[tris(2-carboethyl)phosphine]
[Milli-Q water]
TCEP is located in NCE 436 -20C freezer door (currently the top shelf)
of    have been made and are stored in Teesha's
?. [Preparing Solutions and Materials Needed]
Add to
[CAA]
[2-chloroacetamide]
[Milli-Q water]
CAA is located in NCE 438 chemical room on the "C" shelf
of    have been made and are stored in Teesha's
?. [Preparing Solutions and Materials Needed]
Add to
[NH4HCO3]
[ammonium bicarbonate]
[Milli-Q water]
Upscale or downscale volume needed. Up to 15 mL will be used for each replicate.
?. [Cell Lysis]
Add
[trifluoroethanol]
This step should be done in the fume hood.
1:1 water-TFE acts as a hypotonic aqueous buffer to lyse cells, eliminating the need for detergent. TFE helps protein solubility and denaturation; it readily evaporates, so removing it is easy.
TFE is located in the NCE 435 flammable cabinet.
TFE evaporates fast, so work quickly.
?. [Cell Lysis]
Cool for
[on ice]
?. [Cell Lysis]
Mix the sample for
[with a vortex]
?. [Cell Lysis]
Sonicate the sample for  with the Branson Digital Sonifier 250 at 30% amplitude in pulse mode (pulse ON for 0.2s and pulse OFF for 0.8s) with the tapered micro tip probe.
The Branson Sonifier is the cell distrupter of choice, however, the bench top water bath sonicator can be used for 10 minutes.
?. [Reduction & Alkylation]
Add for a final concentration of
[100 mM TCEP]
?. [Reduction & Alkylation]
Add for a final concentration of
[400 mM CAA]
?. [Reduction & Alkylation]
Incubate in the heating block for at
95 °C
?. [LysC Digestion]
Measure the protein concentration with a NanoDrop (using SCOPES A205 Protein)
?. [LysC Digestion]
Calculate how much LysC is needed for a :
[LysC]
[protein]
?. [LysC Digestion]
Add the calculated amount of LysC to the sample
LysC is in the
?. [LysC Digestion]
Incubate for at
[in the digestion incubator]
?. [Trypsin Digestion]
Calculate how much trypsin is needed for a :
[trypsin]
[protein]
?. [Trypsin Digestion]
Add the calculated amount of trypsin to the sample
Trypsin is in NCE 435 -20 freezer in the door, bottom shelf
?. [Trypsin Digestion]
Incubate for a minimum of at
[in the digestion incubator]
?. [Reduction & Alkylation]
Mix the sample for
[with a vortex]
?. [Reduction & Alkylation]
Mix the sample for
[with a vortex]
?. [Reduction & Alkylation]
Mix the sample for
[with a vortex]
?. [Preparing Solutions and Materials Needed]
[heating block]
?. [Second Trypsin Digestion]
Calculate how much trypsin is needed for a :
[trypsin]
[protein]
A second trypsin digestion is optional. User digression is advised.
?. [Second Trypsin Digestion]
Add the trypsin to the sample
?. [Second Trypsin Digestion]
Incubate for at
[in the digestion incubator]
?. [STAGE Tip]
Proceed to the STAGE tip protocol
?. [LysC Digestion]
Calculate the total amount of protein that is desired to carry forward with the experiment. Keeping in mind that the final peptide concentration will be approximately 10-50% of the protein concentration at this step. Transfer this volume to a new tube. If small enough, transfer to a  .
[lo-bind tube]