Science Highlights

For the First Time, Molecular Imaging Reveals the Localization of Different Forms of Proteins in Biological Tissues

Nano-DESI MSI reveals region-specific differences in proteoform expression

A team of scientists from Purdue University and Northwestern University has developed a novel approach for the spatial localization of different forms of the same protein, called proteoforms. Proteoforms are sensitive markers of the state of the individual cells in tissues, yet it is difficult to characterize their localization in biological systems. The work, which applies a mass spectrometry (MS) imaging technology called nano-DESI MSI to analyze proteoforms from tiny locations on a sample, furnished the first molecular images of these important molecules in tissue samples. Proteoform maps provide the first insights into the biochemical pathways regulating the behavior of living cells in tissues. This study is a proof of concept that the method can be used to study how organisms react to their environment, metabolize and develop in health and disease.

“It is exciting to have the opportunity to map the location of proteoforms in tissues,” said Julia Laskin, William F. and Patty J. Miller Professor of Analytical Chemistry at Purdue University and co-principal investigator (PI) in the study. “Proteoforms are sensitive reporters of the biochemical processes associated with both health and disease.”

In addition to her role at Purdue, Laskin is also PI of the Novel Platform for Quantitative Subcellular Resolution Imaging of Human Tissues Using Mass Spectrometry project, a Transformative Technology Development center in the NIH Common Fund’s Human BioMolecular Atlas Program (HuBMAP). The current report is part of her team’s work as part of HuBMAP to create a sub-cell-level, searchable 3D map that scientists can use to navigate within and study the workings of the human body.

One of the problems in understanding what’s going on within living cells is that a protein can exist in many forms depending on the state of the cell. Cells know how to decorate proteins with different small molecules in response to conditions. A given protein, such as the myelin that insulates the brain’s “wiring,” may have small chemical groups added to it, or have the amino acids that make it up changed in other chemical ways. These changes, biologists have learned, can be critical means by which the body changes the behavior of the protein — and the behavior of the tissues in which that protein can be found. Though scientists have been able to identify proteins in microscopic images for some time, earlier methods weren’t able to tell the difference between the various modifications of a given protein — its proteoforms. So the critical proteoform information had been impossible to see under the microscope.

“This collaboration was born from HuBMAP and is leading to major breakthroughs in technology for localization of the exact forms of proteins in model and human tissues,” said Neil L. Kelleher, Professor of Chemistry, Northwestern University, and co-PI. “Working with the Laskin lab and their deep expertise in localized tissue sampling moved us years ahead and represents a major success case in consortia-led science and technology for spatial biology.”

The team overcame this limitation using a technology called nano-DESI MSI (nanospray desorption electrospray ionization mass spectrometry imaging). This method collects molecules from known locations on a tissue and sends them for the analysis of another powerful tool that scientists had been using for many years: mass spectrometry, or MS. In MS, a molecule in a sample gets electrically charged (ionized) and its mass is determined with high accuracy by moving it through well-characterized electric fields. When different proteoforms of a given protein product are analyzed, they generate distinct “peaks” in MS besides the usual ones for that protein, which help identify how a given protein has been modified.

Nano-DESI MSI works by picking up molecules from tiny portions of a tissue slice and sending them to MS. Moving across the slide in incredibly small increments, the scientists collected and ionized each section of the tissue sample, allowing them to run an MS analysis of each section and identify the proteoforms of the proteins there. This technology allowed them to create microscope images of rat brain tissue showing the different proteoforms of several proteins in each part of the sample. The team reported their results in a cover article in the German Chemical Society’s journal Angewandte Chemie in April 2022.

The HuBMAP Consortium is developing tools to create an open, global atlas of the human body at the cellular level. These tools and maps will be openly available, to accelerate understanding of the relationships between cell and tissue organization and function and human health.

It takes trillions of cells to build a human adult, and how those cells interact, connect, and arrange into tissues has a direct effect on our health. HuBMAP will create the next generation of molecular analysis technologies and computational tools, enabling the generation of foundational 3D tissue maps and construction of an atlas of the function and relationships among cells in the human body. These maps and atlas can lead us to a better understanding of how the relationships among our cells affect our health.

The Human BioMolecular Atlas Program is a consortium composed of diverse research teams funded by the Common Fund at the National Institutes of Health. HuBMAP values secure, open sharing, and collaboration with other consortia and the wider research community.

New Method Makes Genetic Messengers Visible in 3D Within a Single Cell

Using the power of advanced computing and microscope techniques, scientists from the California Institute of Technology and elsewhere have tracked the locations of 10,000 gene products in three dimensions within single cells in the mouse brain. The work offers researchers the ability to track the activities of individual genes, for the first time ever enabling study how the fine distribution of genetic messengers affects the workings of cells and how these add up to the behavior of tissues in the body.

The group employed a method called seqFISH+ to use multiple rounds of imaging and reactions to paint individual mRNA molecules with different sequences of colors under standard microscopes. A kind of barcode, these sequential images allowed them to tell mRNAs—the molecular messengers that carry genes’ instructions throughout the cell—apart from each other at distances that would normally be beyond the ability of the microscope to resolve.

This work was funded by award number 1OT2OD026673-01 through the NIH Common Fund HuBMAP initiative. You can read the team’s April 2019 paper in the journal Nature here.

SABER Method Strengthens Genetic Signals in Microscope

Scientists at Harvard and the University of Washington in Seattle have created a method for amplifying RNA copied from genes within single cells, called SABER (signal amplification by exchange reaction). The new technology promises to detect the RNA output of multiple genes in the same cells with increased sensitivity and speed, and at lower cost.

The research team developed SABER as a way to make mRNA molecules, the genetic messengers in the cell, shine more brightly in a microscope image when labeled by a method called FISH. Standing for fluorescence in situ hybridization, FISH tags mRNA from a specific gene of interest with a lab-made nucleic acid molecule labeled with a fluorescent marker. This in turn lights up the mRNA’s position in the cell. Using SABER, the scientists were able to make FISH signals five to 450 times brighter, detecting mRNAs with much higher efficiency and allowing an automatable “workflow” that could speed study of how gene activity causes differences in cell behavior in health and disease.

This work was funded by award number 1UG3HL145600 through the NIH Common Fund HuBMAP initiative. The team’s June 2019 Nature Methods paper can be read here.

Anchor Strategy Integrates Data from Differing Single-Cell Methods

HuBMAP scientists have developed a new strategy for “anchoring” different types of experimental data that each shed light on only part of a cell’s identity to enable a global view of activities in that cell. Scientists from the New York Genome Center and New York University have used this strategy to knit together data on the production of specific proteins with an atlas of which genes are active in different tissues. The method could transform the depth and breadth of information researchers use to understand organ function at the smallest scales.

While many new methods are allowing scientists to look at cellular function at the smallest levels, these methods do not measure all important factors in a comparable way. In one example highlighting the anchoring strategy, the New York team harmonized signals detected by two experimental methods called scRNA-seq and scATAC-seq. scRNA-seq measures the amount of mRNA molecules—the messengers that relay a gene’s instructions to the machinery that produces proteins—in a single cell. scATAC-seq measures what positions in the protein covering of a cell’s chromosomes have “opened up” to expose the DNA for specific genes to become active. By connecting the two, the scientists were able to leverage the unique characteristics of each method to gain a more comprehensive view of cellular states in the mouse brain. In addition to helping scientists draw information from these biological data accurately, the anchor strategy introduces a novel general framework that enables the transfer of information across distinct single-cell experiments.

This work was funded by award number 1OT2OD026673-01 through the NIH Common Fund HuBMAP initiative. You can find their June 2019 report in the journal Cell here.