Electron impact mass spectrometry in analysis of introduction of stable isotopes of deuterium and carbon-13 into amino acid molecules from biological objects

The work demonstrates the possibility of using electron impact mass spectrometry (EI) on a MB-80A ("Hitachi", Japan) with a double electron focusing for analysis of [ 2H, 13C]amino acid mixtures of L -phenylalanine producing strain of Brevibacterium methylicum and L -leucine-producing strain of Methylobacillus flagellatum, and [ 2H, 13C]amino acids of the total biomass protein isolated while growing the bacterial cells on media containing as a source of stable isotopes [ 2H]methanol, [ 13C] methanol and 2H 2O. For mass-spectrometric analysis of the level of incorporation of stable isotopes 2H and 13C into the molecules of [ 2H, 13C]amino acids the multi-componential mixtures of [ 2H, 13C]amino acids, derived from cultural media and protein hydrolysates after hydrolysis in 6 M 2HСl (3 % phenol) and 2 M Ва(OH) 2 were modified into N-benzyloxycarbonyl-derivatives of [ 2H, 13C]amino acids as well as into the methyl esters of N-dansyl-derivatives of [ 2H, 13C]amino acids, which were separated by RP HPLC on a column with octadecylsilane gel Separon SGX C18. The levels of 2H and 13С enrichment of secreted amino acids and amino acid residues of protein were found to be varied depending on the metabolic pathways of biosynthesis and concentration of 2H- and 13C-labelled substrates in growth media from 20.0 atom % to L -leucine/isoleucine up to 97.5 atom % for L -alanine.