Catch-and-Release Reagents for Broadscale Quantitative Proteomics Analyses

The relative quantification of protein expression levels in different cell samples through the utilization
of stable isotope dilution has become a standard method in the field of proteomics. We describe here
the development of a new reductively cleavable reagent which facilitates the relative quantification of
thousands of proteins from only tens of micrograms of starting protein. The ligand features a novel
disulfide moiety that links biotin and a thiol-reactive entity. The disulfide is stable to reductive conditions
employed during sample labeling but is readily cleaved under mild conditions using tris-(2-carboxyethyl)
phosphine (TCEP). This unique chemical property allows for the facile use of immobilized avidin in a
manner equivalent to the use of conventional reversible-binding affinity resins. Target peptides are
bound to avidin resin, washed rigorously, then cleaved directly from the resin, resulting in simplified
sample handling procedures and reduced nonspecific interactions. Here we demonstrate the stability
of the linker under two different reducing conditions and show how this “catch-and-release (CAR)”
reagent can be used to quantitatively compare protein abundances from two distinct cellular lysates.
Starting with only 40 μg protein from each sample, 1840 individual proteins were identified in a single
experiment. Using in-house software for automated peak integration, 1620 of these proteins were
quantified for differential expression.
Keywords: peptide • proteome • isotope • labeling • quantification • cleavable • cysteine • mass spectrometry (MS)
• chromatography (HPLC) • automated • parallel • avidin • biotin • purification