Lysosomes (pink) are cellular organelles responsible for recycling biomaterial within the cell, shown here interacting with mitochondria (green). Image credit: Felix Kraus/Harper Lab, Harvard Medical School.

New mass spec method gets the ‘nMOST’ out of multiomics data

Biological systems are complex and highly regulated. A single genetic mutation can make all the difference in whether molecules function as part of a fine-tuned machine or if it will impair the system.

To better understand these underlying mechanisms that can lead to disease, scientists need reliable and efficient tools to collect and analyze -omics data — an integrated approach to studying different subsets of biomolecules.  

In new research published in Science Advances, a team of researchers developed a method that uses nanoflow-based multiomic single-shot technology (nMOST) to profile both proteins and lipids simultaneously.

The collaboration was led by Joshua Coon, the Thomas and Margaret Pyle Chair in metabolism at the Morgridge Institute and UW–Madison professor of chemistry, and Wade Harper, department chair of cell biology at Harvard University Medical School.

The Harper Lab studies the molecular pathways involved in neurodegenerative disease, while the Coon Lab develops mass spectrometry and multiomics tools to investigate fundamental questions in cell biology.

“We were delighted to work with the Harper Lab to test and harden our new technologies and couldn’t be more delighted with the outcome,” Coon says.

Felix Kraus

This study used nMOST to assess lipids and proteins associated with lysosomal storage diseases. Lysosomes are organelles involved in the breakdown and recycling of biomolecules within the cell, like biological trash compactors. Mutations in certain proteins can impair lysosomal function, leading to the accumulation of cellular material in the organelle. 

“If the trash system doesn’t work, a lot of stuff accumulates in the cell,” says Felix Kraus, a postdoc in the Harper Lab and co-first author of the paper. “It blocks the recycling and turnover of cellular material, which then causes the cells to be unhappy.”

In addition to understanding how these mutations affect protein function, it is also important to learn how lipids are affected since they are integral to building membranes in the cell.

Yuchen He

Lysosomal storage diseases include mutations that can cause frontotemporal dementia, Niemann-Pick disease type C1 and C2, and are linked with an increased risk of Parkinson’s disease.

“nMOST is like an upgrade or second-generation version of our microflow MOST method,” says co-first author Yuchen He, a former graduate student of the Coon Lab who is now a postdoc with Steve Gygi at Harvard. “It builds on the technology to gain sensitivity and have broader coverage of these two types of biomolecules.”

While the microflow system is already high throughput and robust, the nanoflow method has a flow rate about one-thousand times slower. Less liquid is electrosprayed into the mass spectrometer, using less input material while improving the sensitivity.

Katie Overmyer

“There were several iterations building up to this workflow,” says Katie Overmyer, who works with Coon as the associate director of the Lab for Biomolecular Mass Spectrometry. “There were lots of hurdles to make microflow lipidomics turn into nanoflow lipidomics — some clever mass spec acquisition strategies had to get implemented.”

The team used CRISPR-Cas9 to generate genetic knockouts of 33 mutant proteins that were expanded into HeLa cell lines. It took around 4 weeks of continuous data collection resulting in analysis of 2457 proteins and 1100 lipids.

Most laboratories are equipped with only a single mass spec instrument, and switching the setup to run single proteomics versus lipidomics takes up more time and resources. The team’s goal is to acquire multiomic data as quickly and as comprehensibly as possible, while keeping the system simple and accessible for anyone to use.

“Clinicians are interested in a disease, so they typically focus on one or two mutated genes,” Kraus says. “Our labs have an unbiased, discovery approach — what can we actually learn from the basic biology?”

All the collected data was uploaded to an online repository managed by the Coon Lab. This resource is available to anyone interested in running their own analyses to draw connections between the proteome and lipidome depending on their gene of interest. 

The team is already thinking about ways to improve their methodologies. In addition to proteomics and lipidomics, there are other -omics, with hydrophilic small molecules like amino acids and sugars, not included in this system. One goal is to develop workflows that can make this a possibility.

“If we can include metabolomics, we could have a more comprehensive multiomic single-shot technology,” He says.

Overmyer adds that new mass spec instruments like the Orbitrap Astral analyzer can help acquire data faster — instead of taking a month for acquisition and analysis, it could be as quick as two weeks. 

From the biology side, Kraus notes that many lysosome storage disorders are neuropathologies, and a HeLa cell is not the same as a neuron. Being able to do this work with stem-cell derived neuron cultures, or even patient biopsy samples, they could assess the effects on synapses or neurotransmitter release and trafficking.

“I think if these future implementations come out, we can do more nuanced analysis of lysosomal dysfunction in a neuronal context,” Kraus says. 

He adds: “All our teams with different people working together with their unique strengths and techniques, trying to answer these biology questions — in the end, we hope this is a useful resource for the research community.”