Multitechnique mass-spectrometric approach for the detection of bovine glutathione peroxidase selenoprotein: focus on the selenopeptide

• Published in: Analytical and bioanalytical chemistry Vol. 388; no. 3; pp. 585 - 591
• Main Authors: Ballihaut, Guillaume, Mounicou, Sandra, Lobinski, Ryszard
• Format: Journal Article
• Language: English
• Published: Germany Berlin/Heidelberg : Springer-Verlag 01.06.2007; Springer Nature B.V; Springer Verlag
• Subjects: Alkylation; Analytical chemistry; Animals; Capillary Electrochromatography; Capillary high-performance liquid chromatography-inductively coupled plasma mass spectrometry; Cattle; Chemical Sciences; Chromatography; Chromatography, High Pressure Liquid; Digestion; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Electrospray mass spectrometry; Electrospraying; Enzymolysis; Erythrocytes; Erythrocytes - enzymology; Gel electrophoresis; Glutathione; Glutathione peroxidase; Glutathione Peroxidase - isolation & purification; High performance liquid chromatography; Inductively coupled plasma mass spectrometry; Ionization; Ions; Laser ablation; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Peptides - isolation & purification; Peroxidase; Proteins; Selenium; Selenocysteine; Selenoprotein; Selenoproteins - isolation & purification; Size exclusion chromatography; speciation; Spectrometry, Mass, Electrospray Ionization - methods; Spectrophotometry, Atomic; Glutathione peroxidase; Selenoprotein; Electrospray mass spectrometry; Capillary high-performance liquid chromatography-inductively coupled plasma mass spectrometry; Speciation
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-007-1257-4

Glutathione peroxidase (isolated from bovine erythrocytes) and its behaviour during alkylation and enzymatic digestion were studied by various hyphenated techniques: gel electrophoresis-laser ablation (LA) inductively coupled plasma (ICP) mass spectrometry (MS), size-exclusion liquid chromatography-ICP MS, capillary high-performance liquid chromatography (capHPLC)-ICP MS, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS, electrospray MS, and nanoHPLC-electrospray ionization (ESI) MS/MS. ESI TOF MS and MALDI TOF MS allowed the determination of the molecular mass but could not confirm the presence of selenium in the protein. The purity of the protein with respect to selenium species could be evaluated by LA ICP MS and size-exclusion chromatography (SEC)-ICP MS under denaturating and nondenaturating conditions, respectively. SEC-ICP MS and capHPLC-ICP MS turned out to be valuable techniques to study the enzymolysis efficiency, miscleavage and artefact formation during derivatization and tryptic digestion. For the first time the parallel ICP MS and ESI MS/MS data are reported for the selenocysteine-containing peptide extracted from the gel; capHPLC-ICP MS allowed the sensitive detection of the selenopeptide regardless of the matrix and nanoHPLC-electrospray made possible its identification. [graphic removed]