Rapid screening for S‐adenosylmethionine‐dependent methylation products by enzyme‐transferred isotope patterns analysis

Abstract

We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products. In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution. Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components. To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay. This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis.

Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

“…However, as the mass of the protein increases in larger proteins such as antibodies, which have masses of 150 kDa or more, the −18 Da difference in m / z of native and modified protein may not be distinguishable because of broadening of the isotopic envelope, even with a high‐resolution mass spectrometer. Quantitation may also be compromised as the isotopic envelope of the native protein overlaps with the envelope of the modified protein, and significantly, small amounts of modification cannot be detected with high confidence because of narrow dynamic range and intrinsic noise of mass spectra …”

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5

“…Conceptually, the best approach to distinguish between bona fide deamidation and artifact is to conduct sample preparation—including proteolysis—in 18 O-water, as deamidation is the hydrolysis of an amide (Li et al 2008; Liu et al 2013a; Wan et al 2004; Yao et al 2001). In practice, however, analysis of 18 O-labeled samples can be challenging due to complicated isotopic distributions, as proteolysis also introduces varying degrees of 18 O into the newly generated C-termini (Klaene et al 2014; Liu et al 2012; Liu et al 2014).…”

Discovery and elucidation of unknown protein modifications : brominated coumarin azide as a probe for alkynes

“…We foresee that this tag and workflow will be particularly powerful for more complex systems including detecting protein modifications and drug targets in the cellular milieu [44][45] and systems with undefined chemistry and/or sites for bioconjugation or degradation, and moreover, novel chemistry 46 and biology.…”

“…We will discuss several examples of how scientists first discovered unknown modifications using various analytical techniques and then used various mass spectrometry methodologies to elucidate the novel chemistry, mechanism and sites of the protein modification, to ultimately understand the thought process of the workflows for discovery and elucidation. [45][46][47] In addition to the main peak, there are many peaks appearing in the early eluting (acidic) and late eluting (basic) regions of the chromatogram. For therapeutic development, it is essential to understand the causing factors, chemistry and mechanism of the protein modifications that occur to develop a control strategy that minimizes the impact on product quality.…”

“…This method is similar to SILAC, 85 but both the labeled and unlabeled methione is in the same cell culture sample and the combined CD 3 -methionine and CH 3 -methionine at a 1:1 ratio are taken up by cells. AdoMet was then synthesized from the labeled and unlabeled methionine.…”