January 2010

Mass Spectrometry and Diagnostics: Match Made in Heaven

The measurement of the mass of ionized atomic or molecular species has been around for more than 100 years. For about 50 of them it has come out of the physics lab and into the analytical chemistry realm. More recently, with the establishment of desorption ionization methods mass spectrometry (MS) has taken its first steps into biochemistry research. Some of us think that it will find its true calling in the diagnostic and prognostic world.

In the first wave of biochemical-molecular diagnosis, the techniques that dominated were based on chromatography. Chromatography is powerful, relatively reproducible and well understood. However it is not as sensitive as the sate of the art methods and it may not be specific enough; it is not easy to assign an “unknown” contaminant peak, at least not without complimentary methods, like mass spectrometry. Today the most advanced assays of molecular diagnostics are based on immunoaffinity: antibodies (a special class of proteins) are designed and manufactured that selectively bind to a molecular target of choice (a protein or peptide that carries a specific sequence of aminoacids, an antigen). Antibodies are engineered to “light-up” and be read by the human eye or machine vision, usually using radioactivity or fluorescent techniques. These assays are very sensitive, they can be used for quantitation, they are relatively easy to manage in a clinical setting and can be scaled-up and automated using array methods. They have become extremely successful and large amounts are invested in developing this technology further. However they have some major disadvantages:
Antibody design is time consuming and expensive. It may take weeks or months to develop and fully prepare a new immuno-assay, let alone test it and achieve regulatory approval.

  • Antibody manufacturing (particularly for monoclonal antibodies) is a complicated and very high skill job; it is prone to errors and therefore needs stringent and expensive quality control in all manufacture stages.
  • Immunology assays have limited self-life and logistical issues need to be carefully monitored.
  • By nature, immunoassays are indirect measurements, i.e. antibody manufacturing is off-line compared to assay deployment. The process is therefore prone to human or systemic errors (wrong antibody/assay may be used). Diagnostics industry having the most stringent controls minimizes the likelihood of serious errors, however all additional checks and balances add cost.
  • Although immunoassays can be multiplexed, this also increases the complexity of manufacture and quality assurance.
  • Antibodies are proven to not be as specific as initially expected. Studies have proven that affinity errors can occur and antigens/proteins other than the expected can be captured by an antibody.
  • Antibodies lack sensitivity to coding errors which may result to mutant proteins with similar sequence but very different conformation/biological activity.
  • Antibodies work by attaching themselves to a part of the target protein with a specific aminoacid sequence (the antigen). However the same protein can be found in different post-tranlationally modified states (with different modifications, glycans, lipids and other chemical side chains attached to them, which are critical for their biological function). Antibodies can be fooled by such modifications and generate false negatives.

In case the difference between healthy and disease state is not the amount of a specific protein but its dominant state of modification, antibodies cannot give us the required answer.
Use of immunoassays for targeting a panel of biomarkers (instead of a single biomarker), increases the complexity (cost) proportionally to the amount of biomarkers of the panel.

Mass spectrometry can provide answers to most of the above, while it can be deployed for a wide variety of molecular targets, stand-alone or in combination with immunological methods.

  • MS can analyze with very high sensitivity most types of biological compounds, including proteins, peptides, glycans, lipids and other metabolites.
  • Depending on the mass spectrometric methods used, an MS experiment can lock onto one or several target compounds and monitor their existence or absence (and abundance). Alternatively it can scan all existing signals/compounds, in effect becoming highly multiplexed. Scanning can be carried out in a single experiment, without further development or production costs.
  • Several types of experiments (MS, MSMS, different ionization conditions etc.), providing information from different viewpoints can be programmed to be automatically run using the same sample.
  • Apart from focusing on specific target biomarkers, MS is also capable of looking at the “full picture” of the molecular signature of a sample, without additional effort. A database can then link the result obtained with known states or samples, giving an answer with high statistical significance. An example of this is the microbial ID using MALDI MS, technique which has now been adopted by routine clinical labs.
  • MS, particularly when used in tandem mode (MSMS or MSMSMS, etc.) becomes highly specific, virtually eliminating false positives.
  • Mass spectrometry can be coupled on-line with other ion analysis (ion mobility) or chromatographic (HPLC) or even immunological techniques, to offered unparalleled levels of specificity.
  • Modern mass spectrometric instrumentation is very sensitive and further improvements are expected as instruments become targeted to diagnostic applications.
  • MS experiments are very fast. Direct infusion or laser desorption experiment, generate results in seconds.
  • MS instruments are very reproducible, robust and reliable, while a lot of the analysis steps, including sample preparation, can be automated and deskilled.
  • Although MS instruments are expensive capital equipment, they tend to have very high throughput and low cost per test. Use of mass spectrometric methods in diagnostics is certainly compatible with the trend of making diagnostic test more economical.
  • A single piece of MS hardware may be used for a large variety of assays, utilizing different preparation consumables. This strategy may lead to flexible devices for the large scale clinical labs or targeted point-of-care, even bedside solutions.
  • Because of the rapid and economic results derived from MS based assays, clinical practice may make use of multiple tests per clinical case, to help physicians follow the efficacy of interventions, almost in real time.
  • As MS technology becomes adapted to diagnostics, instruments become smaller, cheaper and easier to use (Fasmatech’s prime strategic goal).

All the above point the way towards a future where mass spectrometry plays a key role in future clinical diagnostics. In our opinion, MS designers in collaboration with the most innovative players in the diagnostic industry will work together to adapt the technology further and address the specific needs of clinical practice. The entry of mass spectrometry in the clinical diagnostics market has started and both fields stand to gain.

Emmanuel Raptakis
CEO, Fasmatech Science and Technology