Isolation and Digestion of Neutrophil Proteins

Published: February 29, 2024

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…

Disclosures

The authors have nothing to disclose.

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

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Cite This Article
Isolation and Digestion of Neutrophil Proteins. J. Vis. Exp. (Pending Publication), e22209, doi: (2024).

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