Profiling Drug‐Protein Interactions by Micro Column Affinity Purification Combined with Label Free Quantification Proteomics^†

Abstract

of main observation and conclusion We developed a method for comprehensively profiling drug-protein interactions using micro-column affinity purification (AP) combined with label-free quantitative (LFQ) proteomics as well as the statistical and bioinformatics analysis. FK506 was used as the experimental model for proof of concept. The true interacting proteins were distinguished from the background proteins by their fold changes of FLQ intensities combined with p-values. Totally 116 FK506 interacting proteins including 5 known target proteins were identified. The method was validated by using the LFQ intensity of the endogenous known drug targets together with statistical analysis. We demonstrated that the micro-column-based affinity purification in combination with LFQ proteomics provides a highly reproducible and robust approach for profiling drug-protein interactions.

Comprehensive Profiling of Rapamycin Interacting Proteins with Multiple Mass Spectrometry-Based Omics Techniques

“…24 Novozym 435 esterase was used to selectively catalyze the esterification of the 40 hydroxyl position of Rap with succinic anhydride. 7 The presence of the PEG3 spacer increases the solubility of Rap to facilitate the pull down experiment.…”

Discovery of AHCY as an Off-Target of Doxorubicin by Integrative Analysis of Photoaffinity Labeling Chemoproteomics and Untargeted Metabolomics

“…The proteins were resuspended in 400 μL of PBS containing 0.5% SDS by sonication. Subsequently, all labeled proteins were pulled down through affinity purification with streptavidin affinity micro-columns . The P2-labeled proteins were eluted with the loading buffer of SDS-PAGE and subjected to SDS-PAGE separation.…”

“…Dox-biotin (10 μM, final concentration) was added to the cell lysates (2 mg/mL) and incubated at 4 °C for 2.5 h to form the drug–protein complex. While DOX/Daun/EPI (20 μM, final concentration) was added to the cell lysates (2 mg/mL) at 4 °C for 2.5 h as competition groups and 10 μM Dox-biotin was added to the competition group, all groups were incubated for another 2.5 h. Subsequently, all labeled proteins were pulled down through affinity purification experiments with streptavidin affinity microcolumns . The Dox-biotin-labeled proteins were eluted with the loading buffer of SDS-PAGE and subjected to SDS-PAGE separation.…”

“…Subsequently, all labeled proteins were pulled down through affinity purification with streptavidin affinity micro-columns. 21 The P2-labeled proteins were eluted with the loading buffer of SDS-PAGE and subjected to SDS-PAGE separation. The SDS-PAGE gels were stained with Coomassie brilliant blue G250, and each gel lane was sliced for in-gel tryptic digestion.…”

“…While DOX/Daun/EPI (20 μM, final concentration) was added to the cell lysates (2 mg/mL) at 4 °C for 2.5 h as competition groups and 10 μM Dox-biotin was added to the competition group, all groups were incubated for another 2.5 h. Subsequently, all labeled proteins were pulled down through affinity purification experiments with streptavidin affinity microcolumns. 21 The Dox-biotin-labeled proteins were eluted with the loading buffer of SDS-PAGE and subjected to SDS-PAGE separation. The proteins were transferred to the PVDF membrane, and Western blot analysis with the antibody of anti-AHCY was performed as described above.…”