Sequencing of phosphoserine‐containing peptides yields normally no identifiable PTH‐derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one‐step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as β‐methylaminoalanine (MAA), S‐ethanolcysteine or S‐ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas‐phase sequencer equipped with an on‐line PTH amino acid analyzer.

No abstract

Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.

No abstract

No abstract