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Editorials |
1 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH
2 Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH
3 Department of Chemistry, Cleveland State University, Cleveland, OH
Mass spectrometry is widely appreciated as a powerful analytical method, characterized by high sensitivity and high information content, that can be used for both the qualitative and quantitative analysis of nearly all types of molecules. In light of this power, I would propose that mass spectrometry is ready for a substantially larger role in routine clinical analyses.
In this issue of Clinical Chemistry, Muddiman and coworkers from the Mayo Clinic present reports in which two levels of mass spectrometry experiments are used to detect and characterize transthyretin variants by accurately measuring the molecular mass of the intact protein isolated from human serum (1)(2). As described by the authors, transthyretin variants can form amyloid in tissues that may ultimately damage those tissues. Currently, diagnosis is generally made after symptoms appear by detecting the amyloid in biopsies of the affected tissue.
The report by Nepomuceno et al. (1) uses a sophisticated dual-source electrospray ionization-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry system to make the transthyretin molecular mass measurement and detect the variant protein. These FT-ICR instruments are the current pinnacle of mass spectrometry performance, vastly exceeding any other instrument type in all relevant issues of merit, including resolution, sensitivity, and mass range. The power of the FT-ICR instrument, and the unique dual-ion source designed and developed by this group (3), is used to carry out an experiment in which the molecular mass of the intact transthyretin protein is measured with an extraordinary mass accuracy of <3 parts per million (i.e., mass accuracy to within 0.05 Da for the 13 761-Da protein). The result of this mass accuracy is illustrated by both the proper detection of several known transthyretin variants and the detection and characterization of a novel double-mutant based on a 2-Da mass difference relative to the wild-type protein. In each case, the mass spectrometry results were verified by DNA sequencing.
The companion report by Bergen et al. (2) describes and tests a combined screening and confirmation strategy that uses a more common mass spectrometry system, a quadrupole mass spectrometer, to triage samples sent for transthyretin characterization. In this strategy, shown in Fig. 1 of the report by Bergen et al., the molecular mass of the transthyretin is initially measured in a liquid chromatography–mass spectrometry experiment. Samples that contain two protein species with different molecular masses or a single species with abnormal molecular mass are referred for follow-up analyses depending on the nature of the result. The most common type of transthyretin variant, the Gly6Ser variant, is detected by a +30 Da mass difference and is followed-up by a rapid but presumptive PCR screen to verify the variant. If the Gly6Ser variant cannot be confirmed by PCR, then DNA sequencing is performed to determine the exact nature of the variant. Similarly, mass differences other than +30 Da are also followed up directly by DNA sequencing. The mass spectrometry experiments described by Bergen et al. (2) were carried out on a quadrupole mass spectrometry system that has far lower resolving power than the FT-ICR instrument used in the report by Nepomuceno et al. (1), but it is also less expensive to buy and easier to operate. The lower resolving power of the quadrupole system means that only variants with protein molecular mass differences >10 Da can be distinguished from normal. The authors correctly point out that current time-of-flight mass spectrometers, which are commonly used for liquid chromatography–mass spectrometry analyses, have an intermediate resolving power (relative to the FT-ICR) that would reduce the detectable mass difference to anything >3 Da. The ultimate result is a strategy in which 
88% of the screened samples are rapidly confirmed as normal and nearly all of the samples are spared DNA sequencing.
Other examples of the use of mass spectrometry for clinical assays can be seen in recent reports in Clinical Chemistry that describe assays for detecting protein variants as indicators of clotting disorders (4), quantification of homocysteine (5) and homocysteine metabolites (6) in serum and urine as cardiovascular risk factors, the simultaneous quantification of plasma estradiol and estrone in fertility and endocrinology testing (7), and the quantification of drugs and metabolites for therapeutic drug monitoring (8).
Considering the power of mass spectrometry that is demonstrated in these reports, the question should arise: Why aren’t mass spectrometers more commonly used for routine analyses in clinical laboratories?
I believe the answer to this question is based in both scientific and nonscientific misconceptions about mass spectrometry, which include the following: (a) mass spectrometry experiments are not amenable to most analytes of interest in clinical assays; (b) mass spectrometry experiments are too slow and difficult to automate for the sample throughput needed in a clinical laboratory; (c) mass spectrometers are expensive to buy and maintain; and (d) the persons who are able to operate mass spectrometers and/or direct a mass spectrometry laboratory are in short supply.
A key to understanding these are misconceptions lies in understanding the impact of electrospray ionization on the current state of mass spectrometry. Ultimately, the impact of electrospray ionization has been so profound that Professor John Fenn received the 2002 Nobel Prize in Chemistry in recognition of its development (9)(10).
The development of electrospray ionization has made mass spectrometry suitable for nearly all classes of biomolecules that would be of interest to the clinical laboratory. Proteins, peptides, amino acids, organic acids and bases, and metabolic products, among others, are all polar molecules that are suited to electrospray ionization. This increased variety of amenable analytes has led to dramatic innovation in the advancement of mass analyzers. Ion trap, linear ion trap, time-of-flight, hybrid quadrupole time-of-flight, and FT-ICR methods have all been pushed forward to provide high-performance systems with different advantages for different types of analyses.
Electrospray ionization also markedly facilitates the combination of mass spectrometry with HPLC. This combination brings both automation and higher sample throughput to the mass spectrometry experiment. The separating power of reversed-phase HPLC makes direct or nearly direct analyses common. In the case of the reports in this issue of Clinical Chemistry (1)(2), the samples were prepared for analysis by a single-step immunoaffinity purification that can be carried out in large numbers using a standard 96-well microtiter plate format. Furthermore, the information content of the mass spectrometry experiment allows relatively rapid HPLC programs be used [16 min in the report by Bergen et al. (2)] without compromising the selectivity of the assay. Other reports noted above describe total run times of 5 min (7), 4 min (8), and 2 min (5), respectively. Finally, HPLC systems can also use automated injector systems that can operate unattended for long periods of time and can even switch between multiple columns to further increase sample throughput.
A possibly underappreciated benefit of the growth of the scientific side of the mass spectrometry experiment is the strength of the instrument market. Mass spectrometers continue to be relatively expensive instruments, costing in the range of $250 000 to $500 000 or more, depending on the type of system. Although these are certainly significant amounts of money, they are not out of line with other types of instrument systems. More important, however, is the fact that the competition generated by the strong instrument market has produced instruments that are better designed, better built, easier to operate, and more reliable than previous generations of mass spectrometers. As a result, one would argue that the ongoing cost of running a mass spectrometer has come down significantly, particularly when one considers the positive effect of higher sample throughput.
The final issue, a shortage of individuals who would be qualified to operate a mass spectrometry laboratory, is perhaps the most difficult. The recent expansion of mass spectrometry has created a corresponding demand for mass spectroscopists. Indeed, at the 2001 and 2002 annual meetings of the American Society for Mass Spectrometry, the number of advertised job openings was nearly two times the number of registered job applicants. Although this gap has lessened considerably at the two most recent meetings, it still appears that the demand for mass spectroscopists exceeds the supply. In this regard, however, these reports from the Muddiman laboratory are also interesting because they remind us that productive advanced mass spectrometry laboratories currently exist in nearly all academic health science centers in the form of both individual investigator research laboratories and/or core service facilities. Therefore, the critical expertise that would be needed to establish clinical facilities is often very close by, and the training of scientists with expertise in biomedical mass spectrometry is ongoing.
In closing, I would note that as a part of a review of clinical applications of mass spectrometry in 1995, I likened the clinical potential of mass spectrometry to the power of magnetic resonance imaging, namely, a sophisticated instrumental technique with the ability to carry out detailed studies of an individual, albeit at the biochemical rather than anatomic level (11). At that time, the role that I expected was as part of a detailed work-up in which nonroutine information about specialized analytes was needed. These reports from the Mayo Clinic demonstrate that ability quite clearly. More importantly, however, the reports cited here also show how various analyses can be made routine using the power of robust and reliable mass spectrometry systems to process relatively large numbers of samples. This role is particularly important because mass spectrometry experiments are currently driving broad initiatives in biomarker discovery (12). These efforts are beginning to identify new analytes that are sensitive and selective indicators of diseases such as heart disease and cancer (13)(14)(15). I would suggest that keeping those analyses on mass spectrometry systems, as opposed to trying to transfer them to other types of assays, is not only possible but will bring the assays to clinical application more rapidly and will retain more of the diagnostic power.
References
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