Pyrylium-based dye and charge tagging in proteomics

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Abstract

Introduction The pyrylium group has been established as a selective reagent for ε-amino groups in proteins. Especially for fluorescence labeling it offers numerous advantages over traditional NHS-ester chemistry such as the small size of the fluorophore leading to a lesser spot shift in 1D- and 2D gel electrophoresis[1, 2]. But even though pyrylium-based fluorescent modifiers have been around for more than a decade, they have not been explored to their full capacity as of yet. In an effort to determine their specificity and potential side products as neither has been evaluated before, peptides containing no, one, and two lysine residues and either one or no cysteine residues were labeled with Lightning Red (py-1, Serva), a dye containing pyrylium functionality [3]. Materials and methods LR and DMF were supplied by Serva Electrophoresis GmbH. [Glu1]-fibrinopeptide B, Substance P and sodium bicarbonate were purchased from Sigma-Aldrich. Transthyretin peptide SKCPLMVK was synthesized by ThermoFisher. MeOH came from Fluka, dithiothreitol (DTT) from Applichem. For labeling, peptides were dissolved in 8 µL NaHCO3 (5mM, pH 9.3) with 10 mM DTT and incubated for 15 min at room temperature. Then 40 µL of MeOH and 5 nmol of LR in 1.97 µL anhydrous DMF were added and the solution was incubated for 2h at 50°C in an ultrasonic bath (yields >30%). Mass spectrometry was used to investigate the reaction products (Q-TOF Premier, Waters Corp.). Samples were supplied either by liquid chromatography (nanoAcquity) or by manual nanospray. Results and discussion Whereas minimal labeling of proteins can easily be achieved using standard protocols, peptide labeling required a longer reaction time (2 h compared to 15 min) as well as a high content of organic solvent (84 % vs 2 - 20 %). Gas phase fragmentation proved both labeling of lysine residues as well of that of the N-terminus though at lower intensities. Labeling of other amino acids could not be detected. In fact the only side reactions found was a rearrangement of the labelled N-terminus with an adjacent glutamic acid in case of GFP as well as nominal additions of 158, 173, 215, 303, and 441 Da in low intensities. As py-1 labelled amines are acid labile and the mobile phase contained 0.1 % formic acid neither decomposition nor gas phase reactions can be ruled out as a source for these modifications. Interestingly, for N-terminally labeled peptides a- and b-type ion series were detected predominantly. Therefore, Lightning Red may also be used for charge tagging of peptides as an alternative to the commonly used quaternary ammonium [4] and phosphonium salts [5]. Such charge tagging improves sequence assignment and sensitivity due to simplification of fragmentation spectra. Conclusively, pyrylium-based modifiers have a high potential in protein and peptide analysis for their specificity, few side reactions, fluorogenic and chromophoric properties (reaction product is colored red), as well as the potential use as a charge tag. References [1] Hoefelschweiger, B. K.; Anal. Biochem.; 2005; 344; 122-129. [2] Meier, R. J.; Anal. Chem.; 2008; 80; 6274-6279. [3] Bayer, M., König, S.; Electrophoresis; 2016; July 27, DOI 10.1002/elps.201600318 [4] Setner, B.; J. Mass Spectrom.; 2014; 49; 995-1001. [5] Liao, P.-C.; J. Am. Soc. Mass Spectrom.; 1997; 8; 501-509.

Speakers from the University of Münster

Bayer, Malte

Interdisciplinary Centre for Clinical Research (IZKF)