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 microg 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.
Gartner
Carlos A
CA
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Elias
Joshua E
JE
Bakalarski
Corey E
CE
Gygi
Steven P
SP
D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D004220:Disulfides / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
D001360:Avidin; D001710:Biotin; D004220:Disulfides; D007202:Indicators and Reagents; D010720:Phosphines; D011506:Proteins; C080938:tris(2-carboxyethyl)phosphine