Clickable report tags for identification of modified peptides by mass spectrometry

Abstract

The identification and quantification of modified peptides are critical for the functional characterization of post‐translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom‐up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click‐chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide‐PTM quantification from various experimental conditions.

Cu-Catalyzed Azide–Alkyne–Thiol Reaction Forms Ubiquitous Background in Chemical Proteomic Studies

“…Finally, to increase the confidence in the identified conjugates and avoid the possibility of artifact formation during the peptide enrichment or elution from streptavidin-coated agarose beads, the 0.1 mM alkyne probe 1−4-supplemented lysates were incubated under CuAAC conditions with an azide S6−S9). 32 This time, all proteins were cleaned up on carboxylatemodified magnetic beads using the single-pot, solid-phaseenhanced sample preparation (SP3) strategy, digested, and submitted to high-resolution whole proteome LC−MS/MS analysis. 33 The release of the reporter ion during peptide fragmentation improves the fidelity of the identification of modified peptides during the search.…”