TITLE: A membrane-enriched preparation of culture samples for mass spectrometry-based proteomics
AUTHORS: Gwendolyn Gallagher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose: </span><span>Preparation of culture samples for mass spectrometry-based proteomics</span></div><div class = "text-block"><span style = "font-weight:bold;">Principle:</span><span> Utilizing a membrane-enrichment method of lysing cells and preparing peptides has yielded higher representation of membrane proteins in our mass spectrometry-based proteomic results.Traditional methods do not adequately extract or digest hydrophobic, transmembrane proteins. Particularly, we can now see full expression patterns of proteorhodopsin, something we could not detect using traditional mass spec proteomics prep.This protocol builds on the work of Molloy (2008</span><span style = "font-style:italic;">) Methods Mol Biol</span><span> (doi:10.1007/978-1-60327-064-9_30), Erde et al. (2014) </span><span style = "font-style:italic;">J. Proteome Res</span><span>. (doi:10.1021/pr4010019), and Waldbauer, et al. (2017) Anal. Chem. (doi: 10.1021/acs.analchem.7b02752).</span></div><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span> Pure culture samples were spun down and flash frozen for proteomics.A carbonate extraction protocol was used for membrane enrichment before eFASP.The membrane fraction was enzymatically digested with both chymotrypsin and trypsin and the cytosolic fraction was digested with just trypsin.These samples were then ready to be processed further by </span><span style = "font-style:italic;">in vitro</span><span>isotopic peptide labeling (diDO-IPTL).</span></div></div>

[STEPS]
?. [Cell Lysis Protocol ]
Cell pellets resuspended in 333 uL wash solution and lysed with QSonica high power sonication (15 min, 1 sec pulse, Ampl 85%)
All samples were previously derived from 4.5 mL pure cultures spun down and flash frozen
?. [Cell Lysis Protocol ]
After sonication, the tubes were centrifuged (2500xg, 8 min) to pellet unlysed debris
?. [Cell Lysis Protocol ]
Supernatant was drawn off and added to 830 uL carbonate extraction solution in a polypropylene microfuge tube
It is very important to check that tubes are compatible with ultracentrifuge
?. [Cell Lysis Protocol ]
Shake samples in polypropylene tubes in 4 C for 1 hour
?. [Cell Lysis Protocol ]
After balancing tubes with additional carbonate extraction solution, membrane pellets were spun down in an ultracentrifuge (115,000 xg, 1 hr)
?. [Cell Lysis Protocol ]
Draw off supernatant and preserve as “cytosolic” fraction and save pellet as “membrane” fraction.
?. [Cytosolic Fraction Prep]
Dilute cytosolic fraction samples in 1:1 in exchange + 20 mM DDT. Additional Eppendorf tubes may be necessary.
?. [Cytosolic Fraction Prep]
Alkylate cysteine thiols with 60 nM iodoacetamide and incubate at room temperature for an hour in the dark.
?. [Membrane Fraction Prep]
Disturb membrane pellets with QSonica high power sonication (10 min, 1 sec pulse, Ampl 85%) in 500 uL LDS buffer + 20 mM DTT.
?. [Membrane Fraction Prep]
Incubate samples at 95 C for 20 minutes
?. [Membrane Fraction Prep]
Incubate samples at 37 C for 30 minutes
?. [Membrane Fraction Prep]
Alkylate cysteine thiols with 60 nM iodoacetamide and incubate at room temperature for an hour in the dark.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Mix 50 uL lysate (membrane or cytosolic fraction) with 400 uL exchange buffer on filter unit.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Spin at 14,000 x g for 10 minutes and discard filtrate
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Repeat steps 13-14 until all lysate is concentrated on filter
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Wash filter unit 3 times with 200 uL exchange buffer by spinning at 14,000 xg for 10 minutes.Discard filtrate each time.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Wash filter 2 times with 200 uL digestion buffer (spin down at 14,000 x g for 10 min)
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Transfer filter unit to passivated collection tube.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Peptide digestion incubation
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
For MEMBRANE fraction: add 100 uL digestion buffer and 2 ug chymotrypsin on filter.Incubate overnight at room temperature (seal tubes with parafilm).After overnight incubation, add 2 ug Trypsin and incubate again overnight at room temperature.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
For CYTOSOLIC fraction: add 100 uL digestion buffer and 2 ug trypsin on filter.Incubate overnight at room temperature (seal tubes with parafilm).
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Centrifuge (14,000 x g for 10 minutes).
KEEP filtrate
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Add 50 uL peptide recovery buffer and centrifuge for 10 minutes at 14,000 x g.
KEEP filtrate
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Repeat step 21
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Add 200 uL ethyl acetate to the filtrate and transfer to LoBind tube.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Add 2.5 uL TFA and vortex gently.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Nearly fill each tube with ethyl acetate, sonicate for 10 s (note: not high power), centrifuge at 14,000 x g for 10 minutes, then discard upper organic layer.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Repeat step 25 two more times.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Place sample tubes (uncovered/caps off) in Dry Block set to 60 C for 5 minutes.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Freeze sample (-80 C), then centrivap to dryness.
?. [Enhanced Filter Aided Sample Preparation (eFASP)]
Dried samples can now be used for IPTL labeling or can be loaded on mass spec in 2% Acetonitrile, 0.1% formic acid as an unlabeled sample.