Tell-tale traces: Mass spectral data of methylated amino acids added to public libraries

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  • Published: Sep 6, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Tell-tale traces: Mass spectral data of methylated amino acids added to public libraries

Blood, sweat and tears

Sometime in the future, a simple pin-prick of blood will be enough to diagnose a plethora of diseases, even if we are not displaying any symptoms. This is happening right now, albeit in a primitive form.

Sometime in the future, a simple pin-prick of blood will be enough to diagnose a plethora of diseases, even if we are not displaying any symptoms. This is happening right now, albeit in a primitive form. Recall, for example, when you gave your arm to a phlebotomist, perhaps when you needed your glucose or thyroid levels tested.

In these cases, scientists look for fluctuations from a ‘norm’ threshold derived from trends in the local population. Anything beyond this threshold will see the patient heading for further tests or treatments. Now, scientists propose to take this principle one step further.

They envisage the usefulness of not only changes in biomolecule levels, but also slight tags to these biomolecules that can flag up an underlying disease. Take the amino acid, glycine, as an example. Scientists have found that this simple amino acid is tagged with a methyl group (H3C) in patients with prostate cancer, offering a way to detect a tumour when it is small and untroublesome. Others too have witnessed the remaining class of amino acids being tagged with one, two or even three methyl groups.

These tell-tale traces—whether amino acids, proteins, nucleic acids, lipids or metabolites—can slosh over into our blood, sweat and tears (and other bodily fluids), which are easily sampled. For Reddy and his team based at the CSIR-Indian Institute of Chemical Technology, the study of metabolites seems most promising. ‘All naturally occurring amino acids are the key intermediates in many metabolic pathways,’ reported Reddy and colleagues in the Journal of Mass Spectrometry. ‘And they are demonstrated as pivotal biomarkers of various diseases’.

Empty shelves

For Reddy and his colleagues, it is the paucity of suitable techniques able to analyse methylated amino acids that stifles their diagnostic potential. Numerous scientists have set out to develop these tests using both GC-MS- and LC-MS-based assays, but ‘no technique has been demonstrated for the analyses of all possible methylated amino acids’.

Accordingly, public mass spectral libraries do not contain data for methylated amino acids, which means that these data points remain buried within their mass spectra, with no spectral standards for mass spectrometrists to compare to.

Spurred on by the opportunity to the fill the bare shelves of the spectral libraries, the Indian scientists set out to develop a GC-MS-based method to define the methylated forms of all twenty amino acids. For this, they first prepared standards of both mono and dimethylated amino acids. They then derivatized these with ethyl chloroformate, which they reasoned would enhance the chromatographic resolution of these volatile analytes. These were then ionized by both chemical and electron-induced methods, and mass analysed by GC-time-of-flight (ToF)-MS.

Selective ion monitoring

To test the usefulness of their novel reference spectral library, Reddy et al. spiked plasma with mono- and di-methylated phenylalanine. This modified amino acid was most suitable, they argued, since it was not detected in their donated plasma and is seldom reported to be blood-borne in metabolite databases.

As before, these standards were derivatized and resolved in order of increasing volatility, but this time the mass spectrometer was trained to selectively monitor for the specific precursor and fragment ions that splinter from mono and dimethylated phenylalanine at minutes 18 and 14. Whilst the neat plasma ‘did not show any peaks’ for the methylated phenylalanines, greater than 93% of the modified amino acids they spiked (this time in water) were recovered in their measurements.

‘The GC-MS method and EI mass spectral data,’ Reddy concludes, ‘would be useful for future metabolomics studies to screen amino acids and methylated amino acids simultaneously.’ The authors now hope to enhance their assay to delve into the lower depths of methylated amino acid detection.

Related Links

Journal of Mass Spectrometry, 2016, 51, 638–650. Reddy et al. Characterization of N-methylated amino acids by GC-MS after ethyl chloroformate derivatization.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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