Review
Application of tandem mass spectrometry to biochemical genetics and newborn screening

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Abstract

Tandem mass spectrometry (MS/MS) has become a key technology in the fields of biochemical genetics and newborn screening. The development of electrospray ionisation (ESI) and associated automation of sample handling and data manipulation have allowed the introduction of expanded newborn screening for disorders which feature accumulation of acylcarnitines and certain amino acids in a number of programs worldwide. In addition, the technique has proven valuable in several areas of biochemical genetics including quantification of carnitine and acylcarnitines, in vitro studies of metabolic pathways (in particular β-oxidation), and diagnosis of peroxisomal and lysosomal disorders. This review covers some of the basic theory of MS/MS and focuses on the practical application of the technique in these two interrelated areas.

Introduction

Mass spectrometry (MS), in the form of gas chromatography-mass spectrometry (GC/MS) using electron impact or chemical ionisation, has played a key role in the discovery of inborn errors of organic acid [1] and fatty acid metabolism, [2] and is also widely used in the investigation of peroxisomal [3] and steroid disorders [4]. The technique requires the separation of components of the sample mixture by capillary gas chromatography and uses the unique fragmentation pattern (or mass spectrum) of the separated compounds in the mass spectrometer for confirmation of identity. It does have serious limitations, however, including laborious sample preparation to make compounds of interest amenable to separation by GC, and long analysis time.

The ability to analyse polar, nonvolatile biological molecules was realised with the introduction of “soft” ionisation techniques such as fast atom bombardment (FAB/MS), thermospray (TSP/MS), and most recently and importantly, electrospray ionisation (ESI/MS), allowing liquid chromatography to interface with mass spectrometry. In these techniques, the mass spectrometer is again acting as a sophisticated detector, capable of identifying the compounds presented to it but still requiring a separation technique.

Tandem mass spectrometry (MS/MS) was developed over 20 years ago [5] and was first applied to biochemical genetics soon afterwards [6]. Although ion trap instruments are capable of multiple MS/MS functions (MSn), the technique is most commonly performed using a triple quadrupole mass spectrometer which, as the name suggests, comprises an ionisation source, three mass filters connected in tandem, and a conventional electron or photomultiplier detector (Fig. 1). Ions produced in the source are selected by the first quadrupole (MS1) for transmission to the second quadrupole, which is designated as the collision cell. In this region, the ions are accelerated and collide with molecules of an inert collision gas (usually argon) and undergo collision-induced dissociation (CID). The fragments produced are transmitted to the final quadrupole (MS2) where they are again selected for transmission to the detector. Ions transmitted by MS1 to the collision cell are called precursor ions (formerly referred to as “parent” ions), and the fragments produced from CID are product ions (formerly “daughter” ions). The MS/MS can be operated to scan for all fragments produced from a single precursor (product ion scan), all precursors producing a single product (precursor ion scan), or to scan both filters a fixed mass apart to select all precursors undergoing a loss of a common noncharged moiety (neutral loss scan). In addition to these scanning modes of operation, the instrument can monitor a number of fixed transitions in a manner analogous to selected ion monitoring in a single quadrupole instrument. This type of operation is often called multiple reaction monitoring (MRM) and is particularly useful to restrict the compounds being detected to those preselected, as opposed to the scanning modes which will detect all compounds undergoing the chosen transition.

Because the first mass filter effectively removes the need for a separation technique prior to introduction of the sample, MS/MS can be used for analysis of complex mixtures with little or no sample clean up. The removal of the time-consuming step of resolving individual components by chromatography allows rapid analysis times, typically around 2–3 min per sample, and the use of stable isotope labelled internal standards allows accurate quantitative analysis. The methodology does have limitations, however: it cannot separate isomers and, in complex biological mixtures, the likelihood of encountering more than one compound with the same molecular weight and producing the same fragments is sometimes high. Therefore, several of the methods, which will be covered later, include a chromatographic separation prior to introduction to the MS/MS.

Section snippets

Carnitine and acylcarnitine analysis

Carnitine is a quaternary ammonium compound which has a key role in the transport of long chain fatty acids into the mitochondria. It also plays a role in detoxification by facilitating recycling of free coenzyme A via the carnitine acyltransferases which catalyse the formation of acylcarnitines from free carnitine and acyl CoA esters. Carnitine esters of short, medium, and long chain fatty acids are present in biological fluids and are found in increased concentrations in plasma and urine

Amino acid analysis

Disorders of amino acid metabolism were some of the earliest inborn errors of metabolism investigated on a large scale. Largely, this reflected the availability of techniques to separate and identify amino acids in body fluids. These techniques included paper, thin layer and ion exchange chromatography, and high voltage electrophoresis, all relying on the production of a coloured product [24] when reacted with ninhydrin. For quantitative analysis of plasma amino acids, ion exchange

Newborn screening

For over 30 years from the initiation of the first newborn screening program based upon Guthrie and Susi's [39] bacterial inhibition assay for phenylalanine in dried blood spots, each time a new disorder was added to a screening program, a new test, often with a completely different technology, was required. Thus, even the most comprehensive programs were limited to screening for a handful of disorders which fulfilled the criteria for a sensitive and specific test for a condition which occurs

Other biochemical genetics applications of MS/MS

Although the focus of tandem mass spectrometry in biochemical genetics has been directed to acylcarnitine and amino acid assays, other applications have been investigated. Diagnosis of peroxisomal disorders is usually performed by demonstrating increased very long chain fatty acids in plasma by gas chromatography/mass spectrometry. Johnson [61] developed an MS/MS method using dimethylaminoethyl esters, applying it to the diagnosis of peroxisomal defects using as little as 5 μl of plasma or

Conclusions

Tandem mass spectrometry has emerged rapidly over the last decade as a key technique in the fields of biochemical genetics and newborn screening. Although the capital cost of equipment is high, its short analysis times and resultant high throughput make it an attractive platform for development of new assays, and the list is likely to expand. One of its most important features is the ability to detect and quantify a very wide range of different metabolites from a single sample preparation and

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