UFC901024 MilliporeAmicon Ultra-15, PLGC Ultracel-PL Membran, 10 kDa

Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.