**2.4 Reduction, alkylation, and ultrafiltration**

Samples were transferred to ULTRA-4 ultrafiltration devices with 10 kDa molecular weight cut-off (Millipore Corporation, Danvers, MA, USA). Proteins larger than 10 kDa are retained in the ultrafiltration device. Centrifugation assisted the ultrafiltration, and the samples were exchanged with fresh UTC until the final DTT concentration was 10 mM. Reduction and alkylation of the samples were performed directly in the ultrafiltration devices using 5 mM tributylphosphine and 50 mM acrylamide as described (Smejkal et al., 2006a). The alkylation reaction was terminated by resuming centrifugation and ultrafiltrative exchange. Bradford Reagent (Sigma-Aldrich Chemicals, St. Louis, MO, USA) was used to measure the protein concentration in each sample.
