Improved measurement of protein synthesis in human subjects using 2H-phenylalanine isotopomers and gas chromatography/mass spectrometry

Preston, T. and Small, A.C. (2010) Improved measurement of protein synthesis in human subjects using 2H-phenylalanine isotopomers and gas chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry, 24(5), pp. 549-553. (doi:10.1002/rcm.4402)

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Abstract

Sensitive methods to measure protein synthetic rate in vivo are required to assess changes in protein expression, especially when comparing healthy with infirm subjects. We have previously applied a ‘flooding dose’ procedure using <sup>2</sup>H<sub>5</sub>-phenylalanine (<sup>2</sup>H<sub>5</sub>-phe) and <sup>2</sup>H<sub>8</sub>-phe isotopomers as tracers, which has proven successful in measuring albumin and fibrinogen synthesis in response to feeding in cancer patients. Using tert-butyldimethylsilyl derivatives, we have observed that <sup>2</sup>H<sub>7</sub>-phe is formed with time in vivo from <sup>2</sup>H<sub>8</sub>-phe, probably during transamination. This increases errors when estimating the fractional synthetic rate (FSR) using the <sup>2</sup>H<sub>8</sub>-phe isotopomer compared with the <sup>2</sup>H<sub>5</sub>-phe isotopomer. We sought to improve this situation by use of an alternative derivative that overcomes this problem whilst also streamlining sample preparation. When using N-ethoxycarbonyltrifluoroethyl (ECTFE) amino acid esters, <sup>2</sup>H<sub>8</sub>-phe is effectively converted into <sup>2</sup>H<sub>7</sub>-phe through fragmentation under electron ionisation (EI), allowing both <sup>2</sup>H<sub>8</sub>-phe and <sup>2</sup>H<sub>7</sub>-phe isotopomers to be measured as a single intense [FORMULA] fragment at 98 Th. To illustrate the improved situation, the mean RMS residual was calculated for all albumin data, for each isotopomer and for each derivative. Albumin-bound Phe was analysed as ECTFE-phe with improved precision, independent of the isotopomer used, confirming that the new derivative is superior.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Preston, Professor Thomas and Small, Mrs Alexandra
Authors: Preston, T., and Small, A.C.
Subjects:G Geography. Anthropology. Recreation > GE Environmental Sciences
College/School:College of Science and Engineering > Scottish Universities Environmental Research Centre
Journal Name:Rapid Communications in Mass Spectrometry
ISSN:0951-4198
ISSN (Online):1097-0231
Published Online:28 January 2010

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