Isolation and Digestion of Neutrophil Proteins

Abstract

This video demonstrates the protein isolation from neutrophils, which is further processed and digested into smaller peptides. These peptides can be used for future analysis.

Protocol

1. Neutrophil Cell Collection and Lysis  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.  Discard the supernatant, and add 15 mL of phosphate-buffered solution (PBS) into the tube. Centrifug…

Representative 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. Max…

Materials

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