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City of Hope Team Develops ICP-MS Method for Multiplexed Protein Quantitation


NEW YORK – Researchers from the City of Hope & Beckman Research Institute have developed an inductively coupled plasma mass spectrometry (ICP-MS) method for multiplexed protein quantitation.

Detailed in a study published this month in Analytical Chemistry, the method uses metal-linked antibodies to simultaneously quantify dozens of proteins with high sensitivity and could prove an effective approach for highly multiplexed measurements of low-abundance proteins, said John Termini, professor of molecular medicine at City of Hope and senior author on the paper.

The approach uses antibodies linked to lanthanide metals to make protein measurements via ICP-MS, essentially repurposing one of the core technologies underpinning Fluidigm's CyTOF mass cytometry system. A sample is treated with these metal-linked antibodies and then the metals are cleaved and quantified by the ICP-MS, with the quantity of metal correlating to the quantity of the antibody to which it was linked and the corresponding protein.

In the CyTOF technology, cells are introduced into the instrument one at a time, which allows researchers to conduct single-cell experiments analogous to flow cytometry but with higher multiplexing capabilities. In their work, the City of Hope researchers simplified the approach, using basic ICP-MS instruments to measure proteins in bulk tissue samples.

Termini noted that City of Hope has CyTOF instruments but that he and his colleagues didn't require the single-cell capabilities for their work.

"We just wanted to look at if we could measure an array of protein responses … in a straightforward way using the metal-labelled antibodies," he said.

Termini said that the researchers were interested in the approach as a potentially more multiplexed and more sensitive method for protein quantitation than the western blots they typically use.

"We've started doing westerns ourselves in the last few years, and they are fine, but if you want to strip [the sample] and re-probe [with a new antibody] it's not so easy," he said. "The technique is pretty robust, but we thought, maybe this can complement that. So we ran side-by-side comparisons with the westerns and generally we found it to be a bit superior."

In fact, according to the study, the researchers found that the ICP-MS assays were an order of magnitude more sensitive than the corresponding western blots.

This has been particularly useful for certain nuclear proteins the group had trouble measuring by western blot. "The enhanced sensitivity afforded by the metal labeling was an advantage," Termini said.

While the CyTOF technology has been around for roughly a decade, initially offered through DVS Sciences, which was then acquired by Fluidigm, Termini said he wasn't aware of any other groups who had used the lanthanide-linked antibodies with ICP-MS for bulk measurements like his team had done.

"I did mention it to the Fluidigm reps when they were installing our [CyTOF] instrument, and they were kind of surprised, they said they hadn't seen that," Termini said.

While Fluidigm would not necessarily see any instrument sales from broader uptake of the technique, it could see more orders for its lanthanide-linked antibodies and its Maxpar kits which allow users to label antibodies of interest. The company declined to comment for the story.

Termini said that one reason the technology had yet to be adapted in this way was the relatively limited use of ICP-MS among biologists.

"Analytical pharmacologists know about ICP-MS," he said. "But it is not typically part of the usual biology armamentarium of analytical approaches."

In the Analytical Chemistry study, the City of Hope team used the approach, which they have termed metal assisted protein quantitation (MAPq), to look at changes in the β-catenin signaling pathway in SW480 human colon adenocarcinoma cells with a small molecule inhibitor. The method observed several changes in expression of proteins within the pathway that were not observable using western blots. Additionally, they were able to collect the data in a single MAPq run compared to 12 western blots.

Termini said that given the advantages, the technique could be useful for quickly looking at multiple groups of proteins, like in signaling pathways, and their responses to different perturbations.

He said that he and his colleagues are particularly interested in the effect of elevated glucose on processes like DNA repair and added that the MAPq approach would allow them to easily monitor changes in expression of large groups of proteins in relevant pathways.

"For example, for nucleotide excision repair there are like 30 proteins involved in the pathway," he said. "We didn't look at them all simultaneously, but you certainly could."

He said his lab was also interested in using the technique to look at, for instance, all the enzymes involved in glycolysis at a single time or all of the enzymes in the tricarboxylic acid (TCA) cycle.

He noted that the complexity of signaling pathways meant researchers often focused their attention on just a few components at a time and that the multiplexing capabilities of the MAPq method could help broaden such analyses.

Termini said that while ICP-MS is not commonly used in biological research, the instruments are widely available and inexpensive. A single quadrupole instrument costs around $80,000 and a triple quad runs around $300,000, he said.

He said the researchers used a single quadrupole ICP-MS instrument for the Analytical Chemistry work but that they were interested in implementing the approach on a triple quad, which could provide a boost in performance.

"We feel that we could probably push the quantitation a bit [with the triple quad], and that is something that we are going to look at," he said.