A Periodate-Cleavable Linker for Functional Proteomics under Slightly Acidic Conditions: Application for the Analysis of Intracellular Aspartic Proteases

Specific elution of captured proteins greatly improves the quality
of proteomic data obtained from pull-downs by avoiding signals from
nonspecific proteins, thus allowing more sensitive identification
of target proteins. This is important in activity-based proteomics
or drug target identification. However, commonly used chemically cleavable
linkers can only be cleaved at close to neutral pH, which prevents
them from being used for proteins binding only at lower pH when no
cross-linking is applied. On the other hand, elution of common acid-cleavable
labels can also coelute proteins bound by ionic interactions. Here,
we report the synthesis and application of a label readily cleavable
by mild oxidation at moderately acidic pH for the noncovalent labeling
and pull-down of intracellular aspartic proteases. Using specific
release, target proteins cathepsin D and napsin A were identified
from human kidney with much higher confidence and without any nontarget
hits.