Short enzymatic reactions in proteomics studies
SKBR3 breast cancer cell extracts were digested with trypsin (or LysC) on short time scales (7min, 15min, 30min, 1h, and 18h), and the results were compared to overnight digestion protocols.
Sample Processing Protocol
SKBR3 cells were cultured in McCoy 5A medium with 10 % FBS, arrested in serum free culture medium for 48h, harvested, lysed by sonication (lysis buffer: Tris 50 mM/pH=8, NaCl 75 mM, urea 8M, DTT 1 mM, protease and phosphatase inhibitors), denatured in the lysis buffer at 56 C for 1h, diluted 10-fold with NH4HCO3 50 mM, and digested with trypsin for 7min, 15min, 30min, 1h and 18h. Replicate measurements for the 7 min and 18h digestions were performed with SKBR3 cells arrested in serum free medium for 24 h, and released with EGF 10 ng/mL for 15 min. These samples were digested with trypsin for 7min or 18h. All samples were cleaned with C18/SCX cartridges, and prepared in a solution of H2O/CH3CN (98:2, v/v)/TFA (0.01 %) for LC-MS/MS analysis. The samples were analyzed by nano-LC (12 cm x 100 um i.d. column, C18 packing/5 um), 200 min gradient, and an LTQ-XL Thermo mass spectrometer, using DDA analysis.
Data Processing Protocol
Proteome Discoverer 1.4, UniProt Homo sapiens database, reviewed, non-redundant (20,199 entries, January 2015); 500-5000 mass range, min/max peptide length 6/144, S/N threshold 1.5, precursor ion tolerance 2 Da, fragment ion tolerance 1 Da, fully tryptic fragments, 4 missed cleavages allowed, no PTMs; FDR stringent 1 %, FDR relaxed 3 %.
Deng J, Julian MH, Lazar IM. Partial Enzymatic Reactions: A Missed Opportunity in Proteomics Research. Rapid Commun Mass Spectrom. 2018 PubMed: 30221418