Isolation and Digestion of Neutrophil Proteins

Protocol

1. Neutrophil Cell Collection and Lysis 

1. Collect neutrophil-differentiated HL60 cells in a 15 mL tube by centrifuging samples at 400 x g for 5 mins. 
   NOTE: Neutrophils should be at a confluency of 3.5 x 10⁵ cells/mL after differentiation.  Harvest approximately five million cells for total proteome extraction. 
2. Discard the supernatant, and add 15 mL of phosphate-buffered solution (PBS) into the tube.
3. Centrifuge samples at 400 x g for 5 min.
4. Repeat steps 1.2 to 1.3. 
   NOTE: Cell samples can be flash frozen with liquid nitrogen and stored at -80°C   after Step 1.4, depending on the experimental time frame. 
5. Add 300 μl of cold 100 mM Tris (hydroxymethyl) aminomethane hydrochloride          (Tris-HCl) pH 8.5 containing a proteinase inhibitor cocktail (PIC) tablet to each sample. 
6. Vortex samples briefly. 
7. Add 1/10 volume of 20% SDS to a final concentration of 2% SDS.
8. To lyse cell samples, probe sonicate samples in an ice bath for 30s on and 30s off for five cycles at 10% power. 
9. Centrifuge samples on a short spin (max speed for 5-10 sec).  
   NOTE: Do not let the cell debris pellet. Use a short spin to collect spray off the tube walls.
10. Transfer samples from the 15 mL tube into a 2 mL tube. 
11. Add 1/10 volume of 1 M stock Dithiothreitol (DTT) to a final concentration of 10 mM DTT. 
12. Vortex samples briefly.
13. Incubate samples for 10 min with shaking at 800 rpm at 95°C.
14. After 10 min, cool samples to room temperature.
    NOTE: Samples can be placed in ice to speed up the cooling process. 
15. Add 1/10 volume of 0.55 mM Iodoacetamide (IAA) to a final concentration of 55 mM IAA.
    NOTE: Perform Step 1.15 in the dark to avoid exposing IAA to light. 
16. Vortex samples briefly.
17. Incubate samples at room temperature for 20 min in the dark. 
18. Add 100% ice-cold acetone at a final concentration of 80% and store samples at      -20 °C overnight. 
    NOTE: Storage at -20 °C can be done for up to two weeks if needed.

2. Digestion of Neutrophil Proteins 

1. Centrifuge samples at 13,500 rpm at 4°C for 10 min to precipitate cell pellets. 
2. Discard supernatant and wash cell pellets with 500 μl of 80% ice-cold acetone.
3. Centrifuge samples at 13,500 rpm at 4°C for 10 min, and remove supernatant. 
4. Repeat steps 2.1 to 2.3. 
5. Air-dry pellets. 
6. Once dry, add 100 μl of 8 M Urea/40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) to cell pellet and water sonicate in a 4°C water bath for 30s on and off, for 15 cycles to resolubilize protein pellets. 
7. Using bovine serum albumin (BSA) tryptophan assay (or equivalent) quantifies protein concentration according to the manufacturer’s instructions. 
8. Add 300 μl of ammonium bicarbonate (ABC) to dilute the sample to a final concentration of 2 M Urea. 
9. To digest 50 μg of protein, add 1:50 Trypsin/LysC (enzyme to protein ratio) to samples on ice—mix samples by gently tapping.
10. Incubate samples at room temperature overnight to perform digestion.  

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Results

Figure 1. Workflow of HL60 cells differentiated into neutrophils for mass spectrometry-based proteomics profiling. Undifferentiated and differentiated (via DMSO chemical stimulation) HL60 cell lines were cultured, collected, and processed for total proteome extraction. Proteins were digested with Trypsin/LysC, and peptides were purified using C18 STAGE-tips, followed by detection by LC-MS/MS. MaxQuant and Perseus were used for data analysis. Figure generated with Biorender.com

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Materials

Name	Company	Catalog Number	Comments
15 ml Falcon Tube	VWR	470225-000
25 mL Serological	Fisher Scientific	14955235
Ammonium Bicarbonate (ABC)	ThermoFisher Scientific	A643-500
Acetone	Sigma Aldrich	34850-1L
BSA assay kit	VWR	CA62408-940
Centrifuge 5810 R	Sigma Aldrich	EP022628188
Dithiothreitol (DTT)	ThermoFisher Scientific	R0861	Prepare 1M stock of DTT aliquots and store at  -20°C
Fetal Bovine Serum (FBS)	ThermoFisher Scientific	12483020	Thaw out, prepare aliquots and stored at -20°C
Heating block
HEPES	Sigma Aldrich	H3375	Prepare 8M Urea/40m HEPES aliquots, store at -20 °C
Iodoacetamide  (IAA)	Fisher Scientific	AAA1471514	Prepare 0.55M stock of IAA aliquots and store at  -20°C. Avoid light exposure
L-glutamine	Fisher Scientific	25030081	Thaw out, prepare aliquots and stored at -20°C
Liquid nitrogen
LoBind Microcentrifuge tubes	Eppendorf	13-698-794
Penicillin Streptomycin, Amphotericin	Sigma Aldrich	A5955	Thaw out, prepare aliquots and stored at -20°C
Neutrophil-differentiated HL60 cells
Phosphate-buffered solution (PBS)	Bioshop	PBS408	Store at room temperature
Probe sonicator	Fisher Scientific	100-132-894
Protease inhibitor cocktail (PIC) tablet	Sigma Aldrich	5892791001	Store at 4°C
RPMI 1640	ThermoFisher Scientific	11875093	Store at 4°C
Sodium Dodecyl Sulphate (SDS)	ThermoFisher Scientific	J18220.A1	prepare 2% SDS solution
SpeedVac Centrifuge	Eppendorf	07-748-15
T75 Flasks	ThermoFisher Scientific	156499
Tris HCL pH 8.5	Fisher Scientific	BP152-1	Store at 4°C
Ultrasonic bath	VWR	97043-960
Urea	Bioshop	URE001.1