Mass Spectrometry

Biography

Biography: Anna Konopka

Abstract

Among numerous technologies to study the proteome, mass spectrometry-based techniques are capable to provide the most accurate and reproducible quantitative data. Proteomic studies can provide two types of data: relative and absolute. For absolute quantification suitable standards are required. Two novel approaches, named RSQ&RIQ (Recombinant Selenium Quantified & Recombinant Isotope-labelled and Quantified) and [Sec-to-Cys]selenoprotein standards, for production of full-length protein standards, which are quantified very accurately, are presented.  In the RSQ&RIQ strategy standard protein production is achieved by cell-free E.coli protein expression system with incorporation of selenium and stable isotope-labelled amino acids (e.g. ¹³C/¹⁵N-labelled arginine and/or lysine) in RSQ and RIQ standard, respectively. RSQ is accurately quantified by ICP MS via selenium detection and is then used as a standard for quantification of RIQ by LC-ESI-MS/MS. The latter then represents the final selenium-free protein standard to be used as internal standard for endogenous protein quantification. RSQ&RIQ methodology is universal approach and can be used for absolute quantification of a variety of proteins. Standard preparation, purification, and characterization are presented for human transferrin and mouse MMP-9 proteins.  In the [Sec-to-Cys]selenoprotein standards strategy the protein production is also achieved by cell-free E.coli system, but DNA template containing the coding sequence of selenoprotein is modified in such a way that all selenocysteine (Sec) codons are site-mutated to cysteine (Cys) codons. During protein synthesis ⁷⁶Se-enriched selenomethionine (⁷⁶SeMet) is introduced allowing for accurate absolute quantification by ICP MS. [Sec-to-Cys]selenoprotein standard production and characterization for human glutathione peroxidase 3 and selenoprotein P are presented.