Resource Articles

Article: How Mass Spectrometry is used for Molecular Genetic Testing


Molecular genetic testingMolecular genetic testing is a critical tool in diagnosing and treating a wide range of genetic conditions. One of the key technologies used in molecular genetic testing is mass spectrometry, which allows scientists to accurately identify and measure the presence and quantity of specific molecules in a sample. In this article, we will explore the role of mass spectrometry in molecular genetic testing and how it is used for many biological applications. We will also discuss how the MassARRAY® System by Agena Bioscience® utilizes mass spectrometry in various applications in the field of genetics and the benefits it offers over alternative techniques.


Understanding MALDI-TOF Technology

The basic stages of mass spectrometry (MS) are ionization, acceleration, deflection, and detection. During a classic MS process, the ionization stage happens when the sample is vaporized and then passes into an ionization chamber resulting in positively charged ions. During the acceleration stage, the positively charged ionization chamber repels the positively charged ions, accelerating them towards three negatively charged slits with progressively decreasing voltage. The speed at which the ions accelerate depends on their mass where lighter ions move faster than heavier ones. In the deflection stage, the stream of positively charged ions is deflected by a magnetic field. The deflection depends on the ion’s mass and charge, with stronger deflection occurring with ions of less mass or with a greater charge. In the final detection stage, the ions are detected by a sensor based on m/z. When an ion hits the detector, it generates an electrical current proportional to its abundance. The signal data are captured as a mass spectrum and the m/z values are analyzed.1,2

With “time of flight” (TOF) MS, the process accelerates ionized samples of different masses and charges to the same energy using an electric field before separating and detecting the ions based on their travel time through a vacuum flight tube of defined length.3 Because ions with different m/z have different initial velocities, and those with less mass and/or with more charge “fly” faster, each hits the detector at a slightly different time. Each impact with the detector generates an electrical signal and those data are calculated and displayed as a mass spectrum of the different ion species in the sample.4 The ionization method was improved with the invention of matrix-assisted laser desorption/ionization (MALDI). The improved ionization method does not lead to a significant loss of sample integrity, which concerns fragile compounds and large molecules.5,6 The combination and the matrix-assisted laser desorption/ionization (MALDI) ionization method and the time of flight (TOF) detection method gives us the MALDI-TOF Mass Spectrometry used today.

Applications for MALDI-TOF Technology

MALDI-TOF MS has a wide variety of uses including the detection of large and small molecules (e.g., polymers, proteins, peptides, nucleic acids, amino acids, lipids, etc.) and a range of practical applications like food allergen quantification, forensic postmortem time interval, epidemiological studies, and detection of antibiotic resistance). The table below outlines some of the major applications and uses for MALDI-TOF. Interest in using MALDI-TOF for molecular diagnostics has increased in the last 4 years. From 2018-2022 “disease diagnosis” is one of the top four keywords that co-occurred with MALDI-TOF in scientific review articles during the years 2018-2022.7  The other three top keywords were “pathogen identification,” “small molecules analysis,” and “nucleic acids analysis.” Two of these top four keywords, “nucleic acids analysis” and “disease diagnosis,” relate to genetic testing, various Agena Bioscience panels, and the MassARRAY’s version of MALDI-TOF analysis.

MALDI-TOF Applications Molecular Testing

How the chemistry of the MassARRAY sets the stage for MALDI-TOF

MALDI-TOF is the technology used for the detection of nucleic acid samples on the MassARRAY System, an automated system by Agena Bioscience. Before analysis, the samples are prepared using specific panel chemistries to detect genetic variations such as single nucleotide polymorphisms (SNPs), insertion/deletions (indels), and copy number variation (CNV). The base chemistry uses multiplexed PCR (Polymerase Chain Reaction) in combination with primer extension. The most common sample preparation method used for SNP (single nucleotide polymorphisms) genotyping with MALDI-TOF MS.8,9 The basic steps in this process are outlined below.


MassARRAY panel chemistries require DNA or RNA extracted as the first input sample. Suitable DNA sample sources include FFPE tissue, fine needle aspirates, buccal cells, or blood plasma, depending on which genetic testing panel is used. The DNA/RNA extraction process can be completed using one of many off-the-shelf extraction kits and systems that are widely available. Extraction isolates the DNA or RNA in the sample and removes other potentially interfering substances. The extracted DNA or RNA is then transferred to a 96- or 384- microtiter plate.

Conventional PCR

After extraction, target DNA or RNA sequence are amplified by conventional PCR using multiplexed panel-specific primers, the resulted amplicons, typically about 60 – 800 bps that include interested sites of genetic variations. Up to 100 primers (for 50 segments) can be amplified in a single reaction using only 5 ng of input sample DNA.

PCR Process


After PCR amplification of the target genes is complete, dNTPs remaining in the amplification reaction mixture are dephosphorylated by an enzyme called shrimp alkaline phosphatase (SAP). This is critical because the nucleotides need to be inactivated so that they cannot be incorporated in the next step, single base extension.

Single Base Extension

Single Base Extension

The single base extension step creates the analyte for the MALDI-TOF analysis. This step uses panel-specific extension primers and termination mixes to generate DNA products with different masses depending on which genetic variants are present. The extension primers anneal next to each variant site being assayed, and the extension reaction incorporates one base that is a dideoxy A, T, C, or G terminator nucleotide. Because each nucleotide has a different mass, extension products with even just one base difference can be distinguished by MS. Different chemistries can alter the ratio of the terminator bases to have higher sensitivity for specific variants. Multiple primers can be multiplexed in a single reaction so long as their masses are different from the other primer extension product combinations in the same well. This results in an extremely high level of multiplexing of up to 50 primers per well of sample. The final product is then diluted with water to ensure there is enough sample volume for analysis.

For example, consider newborns and genetic screening of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene for early diagnosis of cystic fibrosis.10 The CFTR panel helps to determine what nucleotide a newborn has at a single location within a gene associated with cystic fibrosis. An A at that site means a healthy baby and a G means that the baby will likely develop cystic fibrosis. During the single base extension step, a specific extension primer anneals just before that DNA location, and then a single terminator base, either an A or G terminator nucleotide, incorporates into the DNA and stops further extension. Because A has a lower mass than G, an extension product from a healthy baby would have a lower mass than a baby that might develop the disease because of that genetic variant.

Assay Extension Products

How the MassARRAY Utilizes MALDI-TOF and Unique Chemistry to Identify Genetic Variation

The reaction products move onto the MassARRAY System for final sample preparation and the remainder of the analysis workflow. The reaction products are loaded into the Chip Prep Module (CPM), a liquid-handling nano dispenser that finalizes sample preparation for mass spectrometry and transfers the analytes to the MassARRAY Analyzer 4 (MA4), for automated completion of the sample preparation process. The reaction products, or analytes, are desalted and transferred to a silicon chip with a pre-spotted array of an energy-absorbent compound called MALDI matrix. The analyte and matrix co-crystallize upon drying and then the chip is automatically moved to the MA4 for analysis.

SpectroCHIPsSpectroCHIP® is a silicon chip with pre-spotted matrix crystal in 96 and 384 format, where the analytes co-crystallize

Once the Chip Prep Module (CPM) is in the MS, each pad on the chip array is irradiated with laser pulses to desorb and ionize the analyte-matrix co-crystals. The matrix absorbs part of the laser energy which is transferred to the DNA fragments and converts them to the gaseous ions to desorb the analyte. The resulting positively charged DNA molecules accelerate off the pad and up the mass spectrometer flight tube toward an extremely sensitive detector. Molecule separation occurs by time-of-flight so that the lightest ions from the smallest analytes travel fastest and reach the detector first. The time-of-flight data collected via the detector is processed by the integrated MassARRAY Typer software that differentiates the extension products by their mass and panel-specific software that generate reports with a range of information including sample DNA variant calls.

The system can achieve an exceedingly high level of multiplexing as the number of variants that can be analyzed by the system is only limited by the number of different mass and potential variant combinations that can be present in a single well. The MassARRAY can analyze up to 50 targets per well and can use multiple wells per panel resulting in multiplexing capabilities on the lower end of microarrays.

Differences in chemistry are the main driver of the sensitivity of the analysis. For example, the single base extension step can be altered to prefer certain extension products thus increasing the sensitivity for those variants. In certain chemistries, a single type of nucleotide base will be added further increasing the sensitivity to the levels where it can be used for liquid biopsy analysis.

Key advantages of the MassARRAY for Molecular Testing

The MassARRAY system is a powerful tool for molecular testing, offering a number of key advantages over more conventional methods such as real-time PCR and next-generation sequencing (NGS). One of the main advantages of the MassARRAY system is its ability to multiplex much higher than is possible with real-time PCR, which typically uses 6-10 fluorescent channels. This allows for more comprehensive analysis of samples in a single run.

Another advantage of the MassARRAY system is its highly accurate and sensitive detection method, which makes it particularly well-suited for use with samples like liquid biopsy. It can detect low levels of DNA or RNA with high specificity, making it a valuable tool for the analysis of these types of samples.

In addition, the MassARRAY system requires lower sample volumes than other methods, particularly if multiple replicates are used. This makes it more efficient and cost-effective for laboratories. It is also able to work with lower quality DNA or RNA than NGS, as only short amplicons need to remain intact for analysis.

Overall, the MassARRAY system offers the potential for cost savings compared to the expense of NGS, as it does not require the use of expensive fluorescent dyes or bioinformatic analysis. It also has a faster turnaround time, with the ability to go from extracted DNA to a result in a single day. Its easy-to-use workflow, which uses well-known techniques, makes it a valuable tool for molecular testing in a variety of applications.

Different Applications for the MassARRAY

The MassARRAY System is a powerful tool for genetic testing, offering high levels of multiplexing and cost-effective, fast-throughput testing. It has several key applications, including pharmacogenetics (PGx), oncology, and sample identification.

In the field of pharmacogenetics, the MassARRAY System is used to personalize patient care by guiding treatment based on a patient’s genetic information. The Agena VeriDose PGx panels use the MassARRAY system workflow to detect single nucleotide polymorphisms (SNPs) and indels (insertions or deletions) as well as copy number variations (CNVs) that are implicated in drug metabolism pathways and drug toxicity.

Oncology is another key application area for the MassARRAY system, targeting clinically relevant variants in DNA from solid tumor tissue and liquid biopsy samples. These panels can detect somatic variants from heterogeneous samples, and the liquid biopsy panels can analyze circulating tumor DNA (ctDNA) to monitor disease progression.

The MassARRAY System is also useful for sample identification, as sample misidentification and mishandling can lead to incorrect results. The Sample Integrity panels use genetic fingerprinting to verify sample identity.

In addition to these applications, Assays By Agena laboratory services offers custom multiplex assays using markers of interest across many application areas. These custom panels also use the MassARRAY System workflow. Overall, the MassARRAY System is a versatile tool with a wide range of applications in genetic testing, limited only by the ingenuity of those using it.

Watch the MassARRAY System video for more on what sets it apart from other types of molecular testing diagnostic equipment.

1. Everley, R. A., Mott, T. M., Wyatt, S. A., Toney, D. M., and Croley, T. R. (2008). Liquid chromatography/mass spectrometry characterization of Escherichia coli and Shigella species. J. Am. Soc. Mass Spectrom. 19, 1621–1628. doi: 10.1016/j.jasms.2008.07.003 2. Ekström, S., Onnerfjord, P., Nilsson, J., Bengtsson, M., Laurell, T., and Marko-Varga, G. (2000). Integrated microanalytical technology enabling rapid and automated protein identification. Anal. Chem. 72, 286–293. doi: 10.1021/ac990731l 3. De Ranieri, E. When a velocitron meets a reflectron. Nature Methods 4, 8 (2015). doi:10.1038/nmeth.3526 4. Cornish, Timothy & Bryden, Wayne. (1999). Miniature Time-of-Flight Mass Spectrometer for a Field-Portable Biodetection System. Johns Hopkins APL Tech Digest. 20. 5. Dandan Li, Jia Yi, Guobin Han, and Liang Qiao, MALDI-TOF Mass Spectrometry in Clinical Analysis and Research, ACS Measurement Science Au 2022 2 (5), 385-404, DOI: 10.1021/acsmeasuresciau.2c00019 6. Amanda Rae Buchberger, Kellen DeLaney, Jillian Johnson, and Lingjun Li, Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights, Analytical Chemistry 2018 90 (1), 240-265, DOI: 10.1021/acs.analchem.7b04733 7. Dandan Li, Jia Yi, Guobin Han, and Liang Qiao ACS Measurement Science Au 2022 2 (5), 385-404. DOI: 10.1021/acsmeasuresciau.2c00019 8. Vogel N, Schiebel K, Humeny A. Technologies in the Whole-Genome Age: MALDI-TOF-Based Genotyping. Transfus Med Hemother. 2009;36(4):253-262. doi: 10.1159/000225089. Epub 2009 Jul 10. PMID: 21049076; PMCID: PMC2941830. 9. Li, D., Yi, J., Han, G., & Qiao, L. (2022). MALDI-TOF Mass Spectrometry in Clinical Analysis and Research. ACS Measurement Science Au, 2(5), 385-404. 10. Newborn Screening Ontario, Ottawa, ON, NSO Symposium 2015 Poster.