SILAC-Pulse Proteolysis: A Mass Spectrometry-Based Method for Discovery and Cross-Validation in Proteome-Wide Studies of Ligand Binding

  • Jagat Adhikari
  • Michael C. Fitzgerald
Research Article


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.

Graphical Abstract

Key words

Mass spectrometry Proteomics ATP Cyclosporine A Thermodynamics Protein folding Chemical denaturation 



This work was supported by a grant from the National Science Foundation Grant CHE-1308093 (to M.C.F.).

Supplementary material

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Table S1 A-C Summary of experimental conditions used in (A) the CsA-Binding Experiments, (B) the ATP-Binding Experiments, and (C) the control experiments (XLSX 13 kb)
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Table S2-S21A and B Excel spreadsheets containing (A) a listing of the peptide and protein ID’s generated in the Control, CsA-, and ATP-Binding Experiments, and (B) a listing of the peptides and proteins assayed in each experiment (XLSX 736 kb)
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Table S22A-C Summary of the protein hits identified in (A) the control experiments, (B) the ATP-binding experiments, and (C) the CsA-binding experiments (XLSX 23 kb)


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Copyright information

© American Society for Mass Spectrometry 2014

Authors and Affiliations

  1. 1.Department of BiochemistryDuke University Medical CenterDurhamUSA
  2. 2.Department of ChemistryDuke UniversityDurhamUSA

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