Abstract
The non-protein amino acid β-methylamino-L-alanine (BMAA) has been linked to neurodegenerative disease and reported throughout the environment. Proposed mechanisms of bioaccumulation, trophic transfer and chronic toxicity of BMAA rely on the hypothesis of protein misincorporation. Poorly selective methods for BMAA analysis have led to controversy. Here, a recently reported highly selective method for BMAA quantitation using hydrophilic interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS) is expanded to include proteinogenic amino acids from hydrolyzed biological samples. For BMAA quantitation, we present a double spiking isotope dilution approach using D3-BMAA and 13C15N2-BMAA. These methods were applied to study release of BMAA during acid hydrolysis under a variety of conditions, revealing that the majority of BMAA can be extracted along with only a small proportion of protein. A time course hydrolysis of BMAA from mussel tissue was carried out to assess the recovery of BMAA during sample preparation. The majority of BMAA measured by typical methods was released before a significant proportion of protein was hydrolyzed. Little change was observed in protein hydrolysis beyond typical hydrolysis times but the concentration of BMAA increased linearly. These findings demonstrate protein misincorporation is not the predominant form of BMAA in cycad and shellfish.
Highlights
IntroductionOur group has recently reported two highly sensitive and selective new methods for quantitation of BMAA in biological samples, one using hydrophilic interaction liquid chromatography (HILIC)-differential mobility spectrometry-MS/MS (HILIC-DMS-MS/MS)[27] and another using capillary electrophoresis-MS/MS (CE-MS/MS)[28]
The comparison of hydrolysis of these extracts using strong (6 M HCl) and mild (0.02 M HCl) acid showed that BMAA was released from its bound form under conditions not expected to lead to protein hydrolysis
The goal of the current work was to apply the developed hydrophilic interaction liquid chromatography (HILIC)-DMS-MS/MS methodology to study the hydrolytic release of BMAA and proteinogenic amino acids during strong acid hydrolysis of representative shellfish and cycad samples, while using a cyanobacterial reference material (RM) negative for BMAA as a control sample
Summary
Our group has recently reported two highly sensitive and selective new methods for quantitation of BMAA in biological samples, one using HILIC-differential mobility spectrometry-MS/MS (HILIC-DMS-MS/MS)[27] and another using capillary electrophoresis-MS/MS (CE-MS/MS)[28]. Both these methods measure un-derivatized BMAA after total sample hydrolysis with limits of detection of 20 μg/kg dry sample. The recently reported HILIC-DMS-MS/MS method[27] is expanded to include 17 proteogenic amino acids in order to make it useful in the investigation of the protein bound nature of BMAA in biological samples. Through a variety of fractionations and hydrolysis experiments, these methods were applied to a detailed study of the extraction, fractionation and hydrolytic release of BMAA in shellfish and cycad
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