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. Author manuscript; available in PMC: 2016 Sep 9.
Published in final edited form as: J Proteome Res. 2016 Jul 27;15(8):2717–2728. doi: 10.1021/acs.jproteome.6b00245

Table 2.

Comparison of protocols evaluated for protein digestion from FFPE tissue.

Methoda
Procedural Step Urea TFE RapiGest
denaturing
buffer		 6M Urea in 50 mM
NH4HCO3 		50% TFE in 50 mM
NH4HCO3 		0.1% RaipGest in 50
mM NH4HCO3
reducing agent 15 mM TCEP 15 mM TCEP 15 mM TCEP
alkylation agent 40 mM IAM 40 mM IAM 40 mM IAM
dilution buffer 200 mM TRIS 50 mM NH4HCO3 50 mM NH4HCO3
trypsin ratio 1:50, followed by
1:100		 1:50, followed by
1:100		 1:50, followed by
1:100
temperature 37°C 37°C 37°C
duration overnight overnight overnight
a

The digestion protocols were chosen for compatibility with tissue and FFPE samples and included: i) a urea-based protocol used in cell lysates and tissues,41 ii) a TFE protocol previously reported in FFPE protein extraction,29 and iii) a RapiGest method also previously coupled with FFPE.62