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. 2014 Aug 5;86(15):7962-8.
doi: 10.1021/ac501994b. Epub 2014 Jul 21.

Tandem mass spectrometry assays of palmitoyl protein thioesterase 1 and tripeptidyl peptidase activity in dried blood spots for the detection of neuronal ceroid lipofuscinoses in newborns

Mariana Barcenas  1 , Chang Xue, Tatyana Marushchak-Vlaskin, C Ronald Scott, Michael H Gelb, František Tureček
Affiliations

Tandem mass spectrometry assays of palmitoyl protein thioesterase 1 and tripeptidyl peptidase activity in dried blood spots for the detection of neuronal ceroid lipofuscinoses in newborns

Mariana Barcenas et al. Anal Chem. .

Abstract

We report new substrates for quantitative enzyme activity measurements of human palmitoyl protein thioesterase (PPT1) and tripeptidyl peptidase (TPP1) in dried blood spots from newborns using tandem mass spectrometry. Deficiencies in these enzyme activities due to inborn errors of metabolism cause neuronal ceroid lipofuscinoses. The assays use synthetic compounds that were designed to mimic the natural substrates. Incubation produces nanomole quantities of enzymatic products per a blood spot that are quantified by tandem mass spectrometry using synthetic internal standards and selected reaction monitoring. The assays utilize a minimum steps for sample workup and can be run in a duplex format for the detection of neuronal ceroid lipofuscinoses or potentially multiplexed with other mass spectrometry-based assays for newborn screening of lysosomal storage disorders.

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Figures

Figure 1
Figure 1
Tandem mass spectra of (a) (N-Ac-ALLPFGC + H)+ ion at m/z 762, and (b) (N-Ac-AAAPFGC + H)+ ion at m/z 678, both obtained at 25 eV laboratory ion collision energy.
Figure 2
Figure 2
Structures of TPP1 substrate (TPP1-S), enzyme product (TPP1-P), internal standard (TPP1-IS), and the pertinent SRM transitions.
Figure 3
Figure 3
Graphical representation of PPT1 activities in DBS from simplex assays.
Figure 4
Figure 4
Graphical representation of TPP1 activities in DBS from simplex assays.
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