Table 1.

LC/MS analysis for a 5-protein FFPE tissue surrogate

Condition	% protein recovery	Protein Identification by LC/ MS
		% sequence Coverage*
		% False protein ID’s **	Lysozyme	Carbonic Anhydrase	RNAse A	BSA	Myoglobin
Native Protein mixture	100%	3.3 ± 0.6	66%	56%	63%	54%	38%
FFPE; extracted in a water bath	26%	42 ± 4.0	15%	n.d.	7%	n.d.	n.d.
FFPE, extracted with high pressure	96%	7.8 ± 1.5	69%	36%	59%	26%	28%

Multi-protein FFPE tissue surrogates were extracted at 100°C for 30 min followed by 80°C for 2 h in a water bath at atmospheric pressure or with high pressure (40,000 psi) in 50 mM Tris-HCl, 2% (w/v) SDS buffer, pH 4. The extracts were washed extensively, and digested overnight with trypsin at 37°C in 50 mM NH₄HCO₃, pH 7.9 with 20% acetonitrile (v/v) before MS.

^*%Sequence coverage: percent of theoretical tryptic peptides identified by LC/MS/MS.

^**The false identification rate was determined as percentage of proteins incorrectly identified for spectra with scores ≥10.5, for 2 technical replicates. n.d. – none detected.