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# Goal/Experiment:
Preparation of culture samples for mass spectrometry-based proteomics

# A membrane-enriched preparation of culture samples for mass spectrometry-based proteomics

*Gwendolyn Gallagher¹*  
¹Gwendolyn Gallagher [University of Chicago], Jacob Waldbauer [University of Chicago]

Coleman Lab  
Gwendolyn Gallagher

## Abstract

### Purpose:
Preparation of culture samples for mass spectrometry-based proteomics

### Principle:
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) Methods Mol Biol (doi:10.1007/978-1-60327-064-9_30), Erde et al. (2014) J. Proteome Res. (doi:10.1021/pr4010019), and Waldbauer, et al. (2017) Anal. Chem. (doi:10.1021/acs.analchem.7b02752).

### Summary:
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 in vitro isotopic peptide labeling (diDO-IPTL).

## Materials Text

### Equipment
- QSonica high power sonicator
- Optima MAX-XP Beckman Coulter centrifuge
- Regular benchtop centrifuge for Eppendorf tubes
- Labconco CentriVap Cold Trap
- Sonicator bath
- Dry Block
- Incubator (37°C)
- Vortex + Eppendorf tube attachment
- 10, 20, 200, and 1000 μL pipettes
- Tube racks

### Materials
- 10, 20, 200, and 1000 μL tips
- Wash solution
- Carbonate extraction solution
- Polypropylene microfuge tube (Beckman Coulter: 357448)
- Exchange buffer
- 1x LDS buffer
- Dithiothreitol (DTT)
- Iodoacetamide
- Digestion Buffer
- Peptide Recovery Buffer
- Protein LoBind Tube (Eppendorf: 022431081)
- Filtrate tubes and Vivacon 500 (30,000 MWCO HY) concentrator (Sartorius)
- Parafilm
- Ethyl acetate
- Trifluoroacetic acid (TFA)

### Reagents and Solutions
- **Wash Solution:** 50 mM Tris-HCl, pH 7.5
- **Carbonate extraction solution:** 100 mM sodium carbonate
- **Exchange buffer:** 8 M urea, 0.2% (w/v) deoxycholate, 1 M ammonium bicarbonate
- **1x LDS buffer:** 0.666 g Tris HCl, 0.682 g Tris Base, 0.800 g LDS, 0.006 g EDTA, 4 g glycerol in 20 mL milliQ
- **Digestion Buffer:** 50 mM ammonium bicarbonate with 0.2% (w/v) deoxycholate
- **Peptide Recovery Buffer:** 50 mM ammonium bicarbonate

## Cell Lysis Protocol (3h)

1. **Lysis:**
    - Cell pellets resuspended in 333 μL 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

2. **Centrifugation:**
    - After sonication, the tubes were centrifuged (2500×g, 8 min) to pellet unlysed debris

3. **Carbonate Addition:**
    - Supernatant was drawn off and added to 830 μL carbonate extraction solution in a polypropylene microfuge tube
    - It is very important to check that tubes are compatible with ultracentrifuge

4. **Shaking:**
    - Shake samples in polypropylene tubes in 4°C for 1 hour

5. **Membrane Pellet:**
    - After balancing tubes with additional carbonate extraction solution, membrane pellets were spun down in an ultracentrifuge (115,000×g, 1 hr)

6. **Fraction Separation:**
    - Draw off supernatant and preserve as “cytosolic” fraction and save pellet as “membrane” fraction.

## Cytosolic Fraction Prep (1h)

7. **Dilution:**
    - Dilute cytosolic fraction samples in 1:1 in exchange + 20 mM DTT. Additional Eppendorf tubes may be necessary.

8. **Cysteine Alkylation:**
    - Alkylate cysteine thiols with 60 nM iodoacetamide and incubate at room temperature for an hour in the dark.

## Membrane Fraction Prep (3h)

9. **Sonication:**
    - Disturb membrane pellets with QSonica high power sonication (10 min, 1 sec pulse, Ampl 85%) in 500 μL LDS buffer + 20 mM DTT.

10. **Heating:**
    - Incubate samples at 95°C for 20 minutes

11. **Cooling:**
    - Incubate samples at 37°C for 30 minutes

12. **Cysteine Alkylation:**
    - Alkylate cysteine thiols with 60 nM iodoacetamide and incubate at room temperature for an hour in the dark.

## Enhanced Filter Aided Sample Preparation (eFASP) (3d)

13. **Filter Loading:**
    - Mix 50 μL lysate (membrane or cytosolic fraction) with 400 μL exchange buffer on filter unit.

14. **Centrifugation:**
    - Spin at 14,000×g for 10 minutes and discard filtrate

15. **Repeat Steps:**
    - Repeat steps 13-14 until all lysate is concentrated on filter

16. **Washing:** 
    - Wash filter unit 3 times with 200 μL exchange buffer by spinning at 14,000×g for 10 minutes. Discard filtrate each time.

17. **Digestion Buffer:**
    - Wash filter 2 times with 200 μL digestion buffer (spin down at 14,000×g for 10 min)

18. **Transfer:**
    - Transfer filter unit to passivated collection tube.

19. **Peptide Digestion Incubation:**

    **For MEMBRANE fraction:** 
    - Add 100 μL digestion buffer and 2 μg chymotrypsin on filter. Incubate overnight at room temperature (seal tubes with parafilm). After overnight incubation, add 2 μg Trypsin and incubate again overnight at room temperature.

    **For CYTOSOLIC fraction:** 
    - Add 100 μL digestion buffer and 2 μg trypsin on filter. Incubate overnight at room temperature (seal tubes with parafilm).

20. **Centrifugation:**
    - Centrifuge (14,000×g for 10 minutes).
    - **Keep filtrate**

21. **Peptide Recovery:**
    - Add 50 μL peptide recovery buffer and centrifuge for 10 minutes at 14,000×g.
    - **Keep filtrate**

22. **Recovery Replication:**
    - Repeat step 21

23. **Ethyl Acetate:**
    - Add 200 μL ethyl acetate to the filtrate and transfer to LoBind tube.

24. **TFA Addition:**
    - Add 2.5 μL TFA and vortex gently.

25. **Sonication:**
    - Nearly fill each tube with ethyl acetate, sonicate for 10 s (note: not high power), centrifuge at 14,000×g for 10 minutes, then discard upper organic layer.

26. **Replication:**
    - Repeat step 25 two more times.

27. **Dry Block:**
    - Place sample tubes (uncovered/caps off) in Dry Block set to 60°C for 5 minutes.

28. **Freezing:**
    - Freeze sample (-80°C), then centrivap to dryness.

29. **Usage:**
    - 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.

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