title: Susceptibility of the hydroxyl groups in serine and threonine to beta-elimination/Michael addition under commonly used moderately high-temperature conditions.
abstract: The beta-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 beta-elimination has not been critically examined. In this study, two analogous phosphopeptides, KMpSTLSYR and KMSpTLSYR, were subjected to beta-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(2)SO(3) at 37 degrees 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 degrees C for 18 h or for even as short as 1h 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(2)SO(3) at 25 degrees C for 24 h as previously reported. This finding suggests that temperatures above 25 degrees C and excessive alkalinity should be avoided to prevent the beta-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 beta-elimination/Michael addition reaction. The additional modification site(s) may complicate the interpretation of data and lead to an erroneous conclusion.
authors: Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Wei
authors: W
authors: Li
authors: Peter S
authors: PS
authors: Backlund
authors: Robert A
authors: RA
authors: Boykins
authors: Guiyu
authors: G
authors: Wang
authors: Hao-Chia
authors: HC
authors: Chen
mesh_terms: D017665:Hydroxyl Radical / Q000737:chemistry; D013058:Mass Spectrometry; D009943:Organophosphorus Compounds; D010455:Peptides / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013696:Temperature; D013912:Threonine / Q000737:chemistry*
chemical_list: C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D009943:Organophosphorus Compounds; D010455:Peptides; D013912:Threonine; D017665:Hydroxyl Radical; D012694:Serine