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

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. Curr. Protoc. Chem. Biol. 5:45‐66 © 2013 by John Wiley & Sons, Inc.

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…The focus of their work has been the human protein MTases G9a (also known as EuHMT2), GLP1 (also known as EuHMT1), and PRMT1 (for a complete overview, see Table 2). Enzyme‐mediated transalkylation reactions using an azido‐modified AdoMet analogue with engineered G9a and GLP121, 60 and an alkyne‐modified AdoMet analogue with engineered G9a20c and PRMT1 have been demonstrated 20g. Additionally, a selenium‐based SAM analogue with an alkyne linker was effectively employed in transalkyation reactions directed and catalyzed by the native protein MTases GLP1, G9a and SUV39H2 28, 60, 61…”

“…Target proteins are then coupled to a clickable dye, for example, dibenzylcyclooctyne‐coupled dyes, and identified by mass spectrometry (MS) analysis or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE; Figure 3). 21, 60, 61 The approach was used for detecting the substrates of the human protein MTase PRMT3, which preferably resides within the cytoplasm 20a. Interestingly, while the protein MTase localizes in the cytoplasm, 23 % of the modifications were found in the nucleus, thus suggesting a broader role for PRMT3.…”

A journey toward Bioorthogonal Profiling of Protein Methylation inside living cells

“…In addition, its propargyl moiety could be transferred to the targets of PMTs and thus amenable for subsequent target visualization or enrichment upon coupling with the well-established alkyne-azide click chemistry (Figure 3) [36,••37,••38]. While propargyl-SAM was shown to be stable under acidic conditions, this compound is not stable under physiological pH with less desired t 1/2 < 1 min, the time scale that may only be sufficient for a small set of methyltransferases such as SETDB1[•39], NovO, and CouO [•26] to label substrates.…”

“…By taking the advantage of the clickable moieties of Pob-SAM and Ab-SAM coupled with azido/terminal-alkyne-containing fluorescent or biotin probes, we were able to visualize the substrates of PRMT1 and enrich the substrates of G9a/GLP, respectively (Figure 3) [••31,••37,••38,••42]. In addition, PRMT1 Y39FM48G variant and G9a/GLP Y1154A/Y1211A variants are barely active toward SAM, a desired criterion of bioorthogonality for the engineered enzyme-cofactor pairs [••31,••42].…”

“…These results thus demonstrated the merit of Bioorthogonal Profiling of Protein Methylation (BPPM) to reveal methylomes under native cellular settings, which may not be feasible using native PMT-SAM pairs. In contrast to the difficulties to detect or enrich methylated substrates with pan-anti-methyllysine (arginine) antibodies [•13–•14], the azido/terminal-alkyne-handles of the SAM analogues set the amenability for subsequent target visualization and enrichment through the well-established azido alkyne click reaction (Figure 3) [36,••37; ••38]. …”