Side reactions in protein cross‐linking experiments using azide linkers

Dreiling, Alena; König, Simone

doi:10.1002/rcm.6641

Cited by 2 publications

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“…We used LightningRed (LR) , the commercial product of Py‐1, for two‐dimensional comparative fluorescence gel electrophoresis, a technique we have developed for improved comparability of 2D‐PAGE of singular samples . In the course of optimization of experimental parameters, we became interested in the labeling mechanism, also driven by earlier experiences with unexpected side reactions during supposedly straightforward labeling , cross‐linking , or simply sample handling . Although pyrylium ions have been described exclusively as a specific label for primary amino groups throughout the literature, and thiols have been considered as nonreactive , we wondered if reactive cysteine residues, which are notorious for uncontrolled chemistry , would really not be affected.…”

Section: Methodsmentioning

confidence: 99%

Pyrylium‐based dye and charge tagging in proteomics

Bayer

König

2016

Electrophoresis

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The pyrylium group is a selective reagent for ε-amino groups in proteins. In particular, for fluorescence labeling, a number of advantages over traditional N-hydroxysuccinimidyl ester chemistry were recognized such as the rapid prestaining procedure. Here, we have investigated the labeling reaction for the fluorogenic pyrylium dye Py-1 using liquid chromatography coupled to MS with the aim of determining its specificity and possible side products. Peptides containing no, one, and two lysine residue and a choice of no or one cysteine residue were labeled with Py-1 at yields > 30%. Gas phase fragmentation proved both labeling of lysine residues as well as that of the N-terminus also in peptides that contained a lysine residue. Evidence for cysteine labeling was not found, but several other products were detected such as the results of rearrangements with adjacent acidic amino acids. Apart from the use as a fluorogenic label, Py-1 recommends itself for N-terminal charge tagging as alternative to the commonly used quaternary ammonium salts. Predominantly a- and b-type ion series were observed for N-terminally labeled peptides. Further applications include chromophore tagging since the labeled product is not only fluorescent but also colored red.

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