Detailed protein data isolated from single human cells

US researchers have obtained a slew of key information about proteins from single human cells for the first time — the most protein data ever collected from a single mammalian cell, in fact — giving scientists one of their clearest looks yet at the molecular happenings inside a human cell.

Until now, detailed information on proteins inside single cells was hard to come by. The raw ‘data’ — the amount of each protein — in a cell is extraordinarily scant and hard to measure. That’s largely because scientists can’t amplify proteins the way they can genes or other molecular messengers.

That has all changed thanks to the work of scientists at the US Department of Energy’s (DOE) Pacific Northwest National Laboratory (PNNL), who analysed single cells — first from cultured cells and then from the lungs of a human donor — and detected on average more than 650 proteins in each cell — many times more than conventional techniques capture from single cells. Their study has been published in the journal Angewandte Chemie.

The team made the findings thanks to a technology created at the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility located at PNNL. The technology, called nanoPOTS, was developed to measure proteins in a tiny, almost unimaginable amount of material.

“nanoPOTS is like a molecular microscope that allows us to analyse samples that are 500 times smaller than we could see before,” said analytical chemist Ryan Kelly, corresponding author of the paper. “We can identify more proteins in one cell than could previously be identified from a group of hundreds of cells.”

That’s important for a couple of reasons. Some proteins exert immense influence within a cell, perhaps determining whether the cell will live, die, mutate or travel to another part of the body, even when they are at very low levels that are undetectable using today’s methods.

In addition, conventional technologies typically analyse hundreds or thousands of cells, pooling them into one batch for analysis. Those findings represent an average view of what’s happening in that tissue; there is little insight to what’s actually happening in a specific cell. That’s a problem if there’s variability from cell to cell — if some cells are behaving normally while other cells are cancerous, for instance.

In the current study, the team analysed the proteins in a sample of fluid that is less than one-ten-thousandth of a teaspoon. Within that sample, the proteins amounted to just 0.15 ng — more than 10 million times smaller than the weight of a typical mosquito.

But working with such a tiny sample poses significant roadblocks to single-cell analysis. As the material is transferred from one test tube to another, from machine to machine, some of the sample is lost at every stage. And when the original sample amounts to no more than a microscopic droplet, losing even a tiny bit of the sample is catastrophic.

Kelly and his colleague Ying Zhu developed nanoPOTS, which stands for nanodroplet Processing in One pot for Trace Samples, to address this problem of sample loss. The technology is an automated platform for capturing, shunting, testing and measuring tiny amounts of fluid. Keys to the technology include a robot that dispenses the fluid to a location with an accuracy of one millionth of a metre, moving between tiny wells that minimise the amount of surface area onto which proteins might glom.

Within those tiny wells, the scientists ran several steps to isolate the proteins from the rest of the sample. Then, the material was fed into a mass spectrometer that separates out and measures each of hundreds of proteins.

The chip containing multiple wells where proteins from single cells are separated out for further analysis. Image credit: PNNL.

The technology was found to reduce sample losses by more than 99% compared to other technologies, giving scientists enough of the scant material to make meaningful measurements — to tell which proteins are at high levels and which are at low levels. That’s vital information when comparing, for example, brain cells from a person with Alzheimer’s disease to those from a person not affected, or looking at cells that are cancerous compared to nearby cells that are healthy.

The PNNL group is currently developing a protein map of cancerous tumours, with funding from the National Cancer Institute under the Beau Biden Cancer Moonshot Initiative. They have also used nanoPOTS to get a closer look at the proteins involved in the development of type 1 diabetes in the pancreas.

Top image caption: A scientist places a chip into the nanoPOTS system. Once the chip is in place, a robot dispenses fluid into the wells with an accuracy of one-millionth of a metre — a precision necessary when the total fluid sample is no more than one-ten-thousandth of a teaspoon. Image credit: Andrea Starr/PNNL.