Application of <i>Safirinium N</i>-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells

• Main Authors: Joanna Fedorowicz, Magdalena Wierzbicka, Marek Cebrat, Paulina Wiśniewska, Rafał Piątek, Beata Zalewska-Piątek, Zbigniew Szewczuk, Jarosław Sączewski
• Format: Article
• Language: English
• Published: MDPI AG 2020-12-01
• Series: International Journal of Molecular Sciences
• Subjects: fluorescence microscopy; fluorescent probe; H/D exchange; live cell imaging; ionization tag; mass spectrometry
• Online Access: https://www.mdpi.com/1422-0067/21/24/9643
• ISSN: 1661-6596; 1422-0067

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent<i> N</i>–hydroxysuccinimide esters of dihydro-[¹,²,4]triazolo[4,3-<i>a</i>]pyridin-2-ium carboxylates (<i>Safirinium P </i>dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. <i>Safirinium</i> tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom–up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, <i>Safirinium </i>NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as <i>E. coli </i>Top10 bacterial strain.