DCSIMG

Deciphering proteins in detail

Tissue protein analysis is key to diagnose and treat diseases. An innovative technique now enables researchers to measure protein expression in individual cells in a fast and simple way. SCHOTT Nexterion specialty glass substrates help to deliver the full potential of this method known as single-cell western.

Tissue protein analysis is key to diagnose and treat diseases. An innovative technique now enables researchers to measure protein expression in individual cells in a fast and simple way. SCHOTT Nexterion specialty glass substrates help to deliver the full potential of this method known as single-cell western.

Proteins contain the key to understanding diseases like cancer and autoimmune conditions – as well as finding a cure for them. That’s why the analysis of protein expression in tissue plays an important role in diagnostics. “Western blotting” is a well-established analysis method that has been in use for 40 years. Simply put, it involves processing proteins from tissue samples through a gel matrix and sorting them by mass with the help of an electric field. The presorted proteins are transferred from the gel to a more durable membrane overnight, again using an electric field. By later probing the membrane with antibodies that connect to specific proteins and others that react to light, a protein’s concentration, composition or binding behavior can be visualized, giving clues to the presence of a tumor, for example.

“Today, western blotting is used all over the world in almost every biological laboratory,” explains Kelly Gardner, Director of Marketing at ProteinSimple. The drawback: The multiple steps of preparation, probing, incubation, and washing are cumbersome for the researcher or lab personnel, while delivering only averaged results. “Western blotting only provides average protein expressions for the entire sample, which can easily consist of 100,000 cells and more,” says Gardner. “However, it supplies no answer as to whether that averaged measurement applies to individual cells and cell types within mixed samples.” Recent research has shown that particularly the heterogeneous profile of a sample delivers fundamental information for understanding health and disease.”

ProteinSimple and SCHOTT started working together in 2015 to develop a coated substrate that allows for optimal processing in protein analysis tools. From left to right: Kelly Gardner, Eric Jabart (both from ProteinSimple) and Spencer Perry (SCHOTT North America). Photo: Jens Pussel
A customized coated substrate is an essential object used in protein analysis equipment. Photo: SCHOTT

Rethinking conventional Westerns

A few years ago, a bioengineering team at the University of California led by Professor Amy E. Herr, with which Gardner was also affiliated, set out to develop the method and enable fast measurements for individual cells. The core of the concept: a glass chip with a gel layer that allows for roughly 1,000 cells to be singled out and tested for different proteins in one run, and a high-performance device that processes and measures this probe in just 4-6 hours.

“With this single-cell western (or sc western) technique, it becomes possible for researchers to determine which cell is responsible for a tumor and how it behaves,” says Gardner. In 2013, the decision was made to form a start-up, Zephyrus Biosciences, and market the concept. Two years later, the young company was acquired by Minneapolis based Bio-Techne, a holding company for biotech enterprises, which then integrated it into ProteinSimple — the division that develops tools for protein analysis. Today, ProteinSimple markets its Single-Cell Western platform under the brand name Milo. “I always wanted a dog named Milo,” Gardner explains the naming with a smile. “Now it stands for a new trend in the life science tools industry.”

ProteinSimple's Milo™ is the world's first Single-Cell Western platform. It measures protein expression in thousands of single cells in a single run. Photo: Jens Pussel
Researchers can measure protein expression in individual cells in a fast and simple way with the Milo platform. Photo: Jens Pussel

How the Milo platform works

Every Milo kit supplies researchers with chips with a gel layer, as well as a range of buffer solutions that aide in the process. Tiny wells in the gel allow single cells to be captured. With 6,400 and varying in size depending on the chip, these microwells are barely perceptible to the eye. “When the sample suspension is applied most of them remain empty, but about 1,000 cells settle in the microwells, one per well,” says Gardner. This also means that less tissue is needed all in all to perform reliable experiments.

After excess cells are washed off, the chip is placed in the Milo device, where the cells are lysed and proteins are separated in-situ by electrophoresis and immobilized in the gel by UV light. This process takes just a few minutes and eliminates the time-consuming membrane transfer step of the conventional method. To visualize the proteins, researchers can simply use the same validated antibodies as in the standard western method and read out the various protein concentrations on the chip with a conventional microarray scanner. Bonus: “The chips can be archived for up to nine months in case additional testing needs to be done at a later time,” says Gardner.

The Milo kit supplies researchers with chips, made of a glass substrate with a razor-thin surface coating. Photo: Jens Pussel
Up to 6,400 tiny wells in the gel layer allow single cells to be captured. Photo: Jens Pussel

A precise partnership

The chips, 75.6 x 25 millimeters in size and just 1 millimeter thick, are made of a glass substrate with a razor-thin surface coating. The latter plays a key role in this scenario: It ensures that the gel remains in place, so that the microwells can be formed with utmost precision – a process ProteinSimple’s employees have constantly perfected.

In the very beginning, the company used to produce the substrate itself, reports Josh Molho, Director of Engineering at ProteinSimple. However, it soon identified Schott as an ideal partner. “If and only if the substrate and the coating are accurate, the microwells can be accurate as well. Schott worked closely with us to develop precisely the substrate we needed and gave us confidence in quality and capacity as our volume demands grew. Their longstanding experience with specialty glass and coatings was an enormous benefit for us,” states Molho. He adds, “As a start-up, we had to act fast. We needed partners that shared this agility. Working with Schott allowed us to concentrate on our actual work, further marketing of the Milo platform.”

Schott also sees great potential in collaborating with young companies like Zephyrus Biosciences. “The single-cell western technique promises to become one of the next trends in diagnostics,” says Spencer Perry, Sales Manager at SCHOTT North America and the contact person for ProteinSimple. “Biotech start-ups are an essential mechanism in the transfer of scientific discovery from research to advances in the medical field. And, after all, enabling passionate researchers and technologies has always been part of Schott’s DNA.”

ProteinSimple’s development to date suggests that Perry’s assessment is correct. The Milo platform is being used in numerous governmental and private research facilities in the USA, Europe and Asia, reports marketing manager Gardner, primarily in cancer and stem-cell research as well as immunology. And recently, a number of renowned professional journals have published study results obtained using single-cell western analysis, including the science journal “Nature”, considered one of the world’s most respected natural science magazines.

Proteins – building blocks of life

Proteins are found in every cell and are involved in numerous processes in our bodies: They battle infections, provide raw material for the body’s growth and ensure that our muscles are able to help us move about. They also help transport vital substances like oxygen and iron in the blood and, as hormones, convey instructions from the brain to the rest of the body. Our DNA (deoxyribonucleic acid) contains the blueprints for protein composition, which are conveyed to the cell via RNA (ribonucleic acid). And just as DNA is the blueprint, proteins are one of the most important building blocks of life –  and sometimes of death as well. This is because mutations can alter protein activity. Increased protein concentrations may indicate a disease. For example, an elevated concentration of the p53 protein has been found in many tumors.

To visualize the proteins, researchers can read out the various protein concentrations on the chip with a conventional microarray scanner. Photo: Jens Pussel

April 16, 2019

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