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Protein pellets were suspended and denatured in 20 l 7 M urea/2 M thiourea/100 mM TEAB, pH 7.5, reduced with 2 L of 50 mM Tris(2-carboxyethyl) phosphine (TCEP, SCIEX), pH 8.0, at 37 C for 60 min and followed by the addition of 1 L of 200 mM cysteine-blocking reagent (methyl methanethiosulfonate (MMTS, Pierce) for 10 min at room temperature

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