title: SILAC-pulse proteolysis: A mass spectrometry-based method for discovery and cross-validation in proteome-wide studies of ligand binding.
abstract: Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein-ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein-ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method's false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein-ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC, 27708, USA.
authors.forename: Jagat
authors.initials: J
authors.lastname: Adhikari
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013816:Thermodynamics
chemical_list: D005656:Fungal Proteins; D008024:Ligands; D020543:Proteome; D016572:Cyclosporine; D000255:Adenosine Triphosphate