Protein digestion Protein concentration was estimated by BCA assay (Pierce) and the lysates were digested with Lys-C (Wako) in an enzyme/protein ratio of 1:100 (w/w) for 1 h followed by a threefold dilution with 25 mM Tris, pH 8.5 to 2 M GdmCl and further digested overnight with trypsin (Sigma-Aldrich) 1:100 (w:w)
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Although they appropriately used inverse probability of treatment weighting to control for confounding, this approach does not ensure clinical equipoise, limiting causal interpretation because the comparison includes patients who may never have been appropriate candidates for treatment
