Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

Abstract

Abstract. The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATPbinding proteins in the Saccharomyces Genome Database (SGD). The 14 nonannotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…Covalent labeling represents another important approach for monitoring protein conformations and interactions [14][15][16][17][18][19]. Solvent-accessible side chains can be modified by watersoluble reactive species, whereas buried segments are sterically protected.…”

Discovery of Age-Related Protein Folding Stability Differences in the Mouse Brain Proteome

“…In fact, over 90% of the 89 peptide hits were the result of a protein destabilization in the old mouse brain. This is particularly remarkable given that the differentially stabilized protein hits previously identified in an earlier disease state analysis involving cell culture models of breast cancer was almost equally split between stabilizations and destabilizations (41). The ΔC 1/2 values associated with these destabilizations ranged from 0.1 to 0.7 M, which correspond to destabilizations on the order of 0.3 to 1.8 kcal/mol.…”

Thermodynamic Analysis of the Geldanamycin–Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach

“…Hit peptides were identified as those with significant transition midpoint shifts in the (-) and (+) geldanamycin samples. Transition midpoints were assigned using a set of criteria (see Supplemental Information) that we have previously established for the analysis of SPROX data [28,29]. Significant transition midpoint shifts were taken to be those resulting from iTRAQ reporter ion differences in the (-) and (+) geldanamycin samples at or between the transition regions of the two chemical denaturation curves obtained with and without ligand.…”

“…Here we report on the thermodynamic properties of the geldanamycin-Hsp90 complex using the stability of proteins from rates of oxidation (SPROX) technique [26][27][28][29]. The SPROX technique is a mass spectrometry-based approach for measuring the thermodynamic stability of proteins and protein-ligand complexes [26,27,29,30].…”

“…The SPROX technique is a mass spectrometry-based approach for measuring the thermodynamic stability of proteins and protein-ligand complexes [26,27,29,30]. The technique has been previously validated using a series of different model systems, which have included both purified and unpurified proteins and protein-ligand complexes [27,29,30]. Of particular significance to the present study is that the SPROX technique does not require any special labeling of the ligand and that the technique is amenable to the thermodynamic analysis of protein-ligand binding reaction in whole cell lysates.…”