Elsevier

Analytical Biochemistry

Volume 530, 1 August 2017, Pages 31-39
Analytical Biochemistry

Newborn screening by matrix-assisted laser desorption/ionization mass spectrometry based on parylene-matrix chip

https://doi.org/10.1016/j.ab.2017.04.021Get rights and content

Abstract

Newborn screening for diagnosis of phenylketonuria, homocystinuria, and maple syrup urine disease have been conducted by analyzing the concentration of target amino acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) based on parylene-matrix chip. Parylene-matrix chip was applied to MALDI-ToF MS analysis reducing the matrix peaks significantly at low mass-to-charge ratio range (m/z < 500). Reproducibility of inter-spot and intra-spot analyses of amino acids was less than 10%. Methanol extraction was adopted for simple and rapid sample preparation of serum before mass spectrometric analysis showing 13.3 to 45% of extraction efficiency. Calibration curves for diagnosis of neonatal metabolic disorders were obtained by analyzing methanol-extracted serum spiked with target amino acids using MALDI-ToF MS. They showed good linearity (R2 > 0.98) and the LODs were ranging from 9.0 to 22.9 μg/mL. Effect of proteins in serum was estimated by comparing MALDI-ToF mass spectra of amino acids-spiked serum before and after the methanol extraction. Interference of other amino acids on analysis of target analyte was determined to be insignificant. From these results, MALDI-ToF MS based on parylene-matrix chip could be applicable to medical diagnosis of neonatal metabolic disorders.

Introduction

Diverse inborn disorders of metabolism system have been reported [1]. A series of tests, which are known as newborn metabolic screening, could be carried out for medical diagnosis of more common disorders. Left untreated, neonatal metabolic disorders may lead to serious symptoms including seizures, mental retardation, intellectual disability, and even death. However, many newborn disorders could be cured if they are recognized and treated at early stage [2]. It was reported that 10 million newborns are tested for inborn metabolic disorders annually [3]. Among various neonatal metabolic disorders, phenylketonuria (PKU) [4], homocystinuria (HCU) [4], [5], [6], and maple syrup urine disease (MSUD) [7], [8], [9] are the most common disorders. Newborn screening was performed with classical methods including Guthrie test, substituting bacterial inhibition assay, and enzyme immunoassay. Recently liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been applied to newborn screening by simultaneous quantification of amino acids in blood [10], [11](a). PKU has been generally diagnosed by the phenylalanine-to-tyrosine ratio at cut-off [11b], whereas MSUD has been screened by the concentration ratio of branched chain amino acid (BCAA) to unbranched chain amino acid [11c]. HCU has been diagnosed by the concentration of methionine in blood [4], [5], [6].

MALDI-ToF MS has been widely applied for the detection of biomolecules owing to its advantages such as easy sample preparation, wide detection range, high sensitivity, and short analysis time [12]. Compared to LC-MS/MS which is a typically used technique for newborn screening, MALDI-ToF MS requires small volume of sample. Moreover no waste solvent is produced after detection. However characterizing small molecules with MALDI-ToF MS leads to many noise peaks related to organic matrix molecules at low mass-to-charge ratio range (m/z < 500) [13]. Quantitative analysis by this technique was also reported to be very difficult because inhomogeneous crystals of sample and matrix result in poor reproducibility of MALDI-ToF MS. Recently we reported that MALDI-ToF MS based on parylene-matrix chip could be used for analysis of small molecules by reducing noise peaks from matrix molecules at low mass-to-charge ratio range [14]. Quantitative analysis was also demonstrated to be possible for four classes of amino acids: polar, nonpolar, acidic, and basic. Owing to its high speed, less sample consumption, high sensitivity, and capability of quantitative analysis with less matrix-related noise peaks at low mass-to-charge ratio range, MALDI-ToF MS based on parylene-matrix chip was expected to be effective for medical diagnosis of neonatal disorders. In this work, feasibility of parylene-matrix chip for diagnosis of PKU, HCU, and MSUD was demonstrated by quantitative analysis of four amino acids (phenylalanine, methionine, leucine, and valine). Furthermore, very rapid methanol extraction was employed for sample preparation of serum before mass spectrometric analysis.

Section snippets

Materials

Phenylalanine, methionine, leucine, valine, α-cyano-4-hydroxycinnamic acid (CHCA), trifluoroacetic acid (TFA), and methanol, formic acid were purchased from Sigma-Aldrich Korea Co. (Seoul, Korea). Acetonitrile was from J.T. Baker (Philipsburg, NJ, USA). Parylene-N precursor was purchased from Femto Science (Hwasung, Korea). Stainless steel plates were purchased from Goodfellow Cambridge Ltd. (Huntingdon, UK). Doubly distilled deionized water (18 MΩ cm) was prepared using Milli-Q system

Detection of amino acids by MALDI-ToF MS based on parylene-matrix chip

In conventional MALDI-ToF MS, organic matrices are used for ionization of sample molecules. Matrix molecules are known to be fragmented and ionized by absorption of UV laser radiation, and produce strong noises at low mass-to-charge ratio range. Usually, mass peaks from matrix are not reproducible. Analyzing small molecules by MALDI-ToF MS, it is nearly impossible to discriminate the signal peaks of analyte with matrix-related peaks. Recently parylene-matrix chip was used to diminish the mass

Conclusions

Simple and rapid newborn screening tests for PKU, HCU, and MSUD were developed by quantitative measurement of phenylalanine, methionine, leucine, and valine using MALDI-ToF MS based on parylene-matrix chip. Parylene-matrix chip showed a surprising performance in MALDI-ToF MS reducing the background noise dramatically from the matrix and enabled the analysis of small molecules. Amino acids could be analyzed quantitatively at around their specific cut-off levels, with low LOD values using MALDI

Acknowledgments

This research was supported by the National Research Foundation funded by the Korean Government (2014M3A9E5073818), by the Korea Small and Medium Business Administration (S2220656) and by Korea Institute of Science and Technology (KIST) Institutional Program (2E26990).

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1

Jo-Il Kim and Joo-Yoon Noh contributed equally as first author.

2

Current affiliation: Institute of Pharmaceutical and Medical Chemistry, University of Muenster, Muenster, Germany.

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