Work

Bioorthogonal Profiling of Protein Methylation (BPPM) Using an Azido Analog of S‐Adenosyl‐ L ‐Methionine

Year: 2013

Type: article

Abstract: Protein methyltransferases (PMTs) utilize S-adenosyl-L-methionine (SAM) as a cofactor and transfer its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes, from transcriptional activation/silencing to cell cycle regulation. This article describes recently developed technology based on protein engineering in tandem with SAM analog cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analog; (2) generating a proteome containing the engineered PMT; (3) visualizing the proteome-wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag; and finally (4) pulling down the proteome-wide substrates for mass spectrometric analysis.

Source: Current Protocols in Chemical Biology

DOI: 10.1002/9780470559277.ch120240

Authors: Gil Blum, Kabirul Islam, Minkui Luo

Institution: Memorial Sloan Kettering Cancer Center

Language: English

Topic: Click Chemistry and Applications

Subfield: Organic Chemistry

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: National Institutes of Health, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke

Awards: Enzyme-engineering Approaches to Dissect Protein Methylation Profiles, Profile substrates and inhibitors of protein lysine methyltransferase, Developing High Throughput Assay to Identify Protein Methyltransferase Inhibitors