Susceptibility of the hydroxyl groups in serine and threonine to β-elimination/Michael addition under commonly used moderately high-temperature conditions

Abstract

The β-elimination/Michael addition reaction has been employed for the modification of O-acylated and phosphorylated Ser and Thr residues in a variety of derivatives. The modified Ser and Thr can be analyzed by amino acid composition analysis, N-terminal Edman degradation sequence analysis, and tandem mass spectrometric sequencing which generally allows the identification and localization of the phosphorylation or glycosylation sites. However, the reactivity of the free hydroxyl group on serine and threonine by sodium hydroxide-induced β-elimination has not been critically examined. In this study, two analogous phosphopeptides, KMpSTLSYR and KMSpTLSYR, were subjected to β-elimination under the widely used conditions previously reported, followed by sulfite or ethanethiol addition. After treatment of the phosphopeptides in 0.1 N NaOH/0.6 M Na 2SO 3 at 37 °C for 24 h, matrix-assisted laser desorption ionization-time of flight mass spectrometric analyses of the products revealed an appreciable mass peak with an additional observed mass of 64 compared to the expected mass from the conversion of phosphate to sulfite. Similarly, treatment of the phosphopeptides in 0.52 N NaOH/1.36 M ethanethiol at 50 °C for 18 h or for even as short as 1 h also yielded additional 44 mass of ethylthiogroup in excess of the expected mass for the modified phosphopeptide. Electrospray ionization tandem mass spectrometric analysis confirms that the modification occurred on the hydroxyl group of Ser and Thr in addition to P-Ser and P-Thr. On the other hand, modification on the free hydroxyl group of Ser or Thr was not detected under the mild condition of 0.1 N NaOH/0.6 M Na 2SO 3 at 25 °C for 24 h as previously reported (Li et al., Anal. Chem. 74:5701-5710, 2002). This finding suggests that temperatures above 25 °C and excessive alkalinity should be avoided to prevent the β-elimination of the hydroxyl group of Ser and Thr in peptides. This is of particular concern when employing highly sensitive tandem mass spectrometric methods for the identification and localization of Ser and Thr as modification sites by the β-elimination/Michael addition reaction. The additional modification site(s) may complicate the interpretation of data and lead to an erroneous conclusion.