Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry.
In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.
Karnaukhova
E
E
Department of Organic Chemistry, Leiden University, The Netherlands.
Niessen
W M
WM
Tjaden
U R
UR
Raap
J
J
Lugtenburg
J
J
van der Greef
J
J
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D006217:Halobacterium / Q000378:metabolism; D017879:Isothiocyanates; D007553:Isotope Labeling; D011506:Proteins / Q000032:analysis*; D013861:Thiocyanates; D014364:Tryptophan / Q000032:analysis*
D000596:Amino Acids; D017879:Isothiocyanates; D011506:Proteins; D013861:Thiocyanates; C005441:phenylisothiocyanate; D014364:Tryptophan; D003903:Deuterium