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. 2018 Jan 8;8(1):117.
doi: 10.1038/s41598-017-18392-w.

Differential Mobility-Mass Spectrometry Double Spike Isotope Dilution Study of Release of β-Methylaminoalanine and Proteinogenic Amino Acids During Biological Sample Hydrolysis

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Free PMC article

Differential Mobility-Mass Spectrometry Double Spike Isotope Dilution Study of Release of β-Methylaminoalanine and Proteinogenic Amino Acids During Biological Sample Hydrolysis

Daniel G Beach et al. Sci Rep. .
Free PMC article

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.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Summary of sample preparation workflows used in this study.
Figure 2
Figure 2
Full scan (A,B,C) and collision induced dissociation (D,E,F) spectra of protonated BMAA (A,D), D3-BMAA (B,E) and 13C15N2-BMAA (C,F). Product ions used in SRM experiment are shown in bold. Spectra were collected by infusion ESI-DMS-MS(/MS) using a collision energy spread of 15 V to 20 V.
Figure 3
Figure 3
HILIC-DMS-MS/MS (A) and HILIC-MS/MS (B) chromatograms of naturally occurring BMAA, 125 nM spiked 13C15N2-BMAA and 125 nM spiked D3-BMAA in hydrolyzed mussel tissue RM.
Figure 4
Figure 4
Separation of 17 proteinogenic amino acids in a mixed standard solution by ESI-DMS-MS/MS.
Figure 5
Figure 5
HILIC-DMS-MS/MS analysis of proteinogenic amino acids in a 500 fold dilution of a mussel tissue RM hydrolyzed for 12 h at 100 °C. All plots are normalized to the intensity of phenylalanine.
Figure 6
Figure 6
Distribution of BMAA (A) and protein as measured by hydrolytic release of proline (B) between free (unhydrolyzed), soluble (hydrolyzed) and total (hydrolyzed) cycad seed, mussel tissue RM and lobster samples. Error bars show standard deviations of triplicate sample preparations.
Figure 7
Figure 7
Release of BMAA (A), proline (B) and 13C15N2-BMAA internal standard stability (C) during strong acid hydrolysis of a mussel tissue RM. BMAA concentration determined by double isotope dilution using 13C15N2-BMAA spiked before hydrolysis. Error bars represent standard deviation of multiple sample preparations (2 ≤ N ≤ 6). Insets expand results from the first 12 h of the experiment.

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