A New Approach Improves Signal Detection in Mass Cytometry

Xiaokang Lun, currently a systems biologist at Harvard University, used mass cytometry in his graduate research to study intracellular signaling. However, while this method can query up to 50 unique proteins with limited overlap using metal-tagged antibodies, it requires high signal production for detection, complicating studying low-abundance proteins. As a postdoctoral researcher in Peng Yin’s lab, Lun and his colleagues developed a new approach using DNA molecules to enhance the signal from these targets. The technique, amplification by cyclic extension (ACE), is suitable for in vitro mass cytometry analysis as well as imaging mass cytometry (IMC) studies.¹

What motivated you to develop the ACE technique?

One of the limitations of mass cytometry is its low sensitivity. To reach the detection limit, the target needs multiple copies of the antibody tag bound, which makes studying low-abundance targets, either whole proteins or post-translational modification (PTM) sites, difficult. Our group previously developed a different method to amplify these signals from cells and tissues in situ, but it was not effective for in vitro mass cytometry samples.²

What happens in this approach?

Unlike in traditional mass cytometry, where the metal tag is bound directly to the antibody that will bind to the target, we added short DNA polymers to the antibody that extend over a series of amplification steps. We bound a metal-conjugated detector polymer to an oligonucleotide strand that recognized these extender sequences to tag these amplified DNA polymers. This process amplified the specific target signal more than 500-fold.

The DNA extenders bound to the target-specific antibody provide more binding sites for the ultimate detector, so this overcomes the signal to background problem of low-abundance targets. Not only could this help in identifying low-abundance targets, like PTMs, at the single-cell level, but it can also extend mass cytometry to studying smaller cells that traditional methods couldn’t detect. We also showed that ACE can be applied to IMC and overcomes limited antibody signal that occurs with traditional IMC while still offering the advantages of mass cytometry that avoid autofluorescence in standard fluorescence microscopy.

Schematic of the amplification by cyclic extension technique (ACE) for mass cytometry.

To increase the signal detection of low-abundance proteins by mass cytometry researchers developed a method called amplification by cyclic extension (ACE). They bound unique DNA oligonucleotide sequences to target-specific antibodies, incubated the sample with the DNA-bound antibodies, and removed unbound proteins with washing (1). Using an oligonucleotide that contains two consecutive sequences complementary to those on the DNA-bound antibodies and a polymerase, the researchers performed multiple rounds of amplification to extend the DNA strand bound to the antibody (2). After amplification, the researchers added detector oligonucleotides bound to a metal ion. The sequences of these detectors were complementary to the DNA-bound antibodies. The detector also contained a nucleic acid that undergoes crosslinking to an opposite adjacent base when exposed to ultraviolet light (3). The metal-ion-bound amplified DNA antibody tag provided improved signal for both suspended mass cytometry and imaging mass cytometry samples (4).

Imran Chowdhury, adapted from Lun X, et al.

What were some of the challenges in developing ACE?

One challenge was preventing the DNA extensions from denaturing during a 200-degree Celsius heating step to vaporize the samples. We incorporated a crosslinking step and that improved the stability of the DNA in this step. Additionally, we encountered an issue in the sample introduction step. This turned out to be because the tubing on the instrument was made of silica, which binds to the DNA. Replacing this tubing with non-DNA binding plastic resolved this problem.

What are some future directions for this technique in your lab?

One future project in the lab is to simplify the antibody conjugation protocol that we currently use so that it is more accessible. We are also interested in applying this signal amplification into other methods, like enzyme-linked immunosorbent assays (ELISAs) and western blots.

Disclosure of Conflicts of Interest: Xiaokang Lun, Peng Yin, and another study coauthor have applied for a patent related to the ACE method.