Feature: Top down proteomics

By Susan Williamson

Read part I of top down proteomics.

Previously, the field of top down proteomics has lagged behind the bottom up approach in terms of the throughput that can be achieved.

This was because the three basic parts to the whole protein analysis procedure – sample processing, mass spectrometry and bioinformatics – were not adequately developed for detecting intact proteins.

There has been a big push in Professor Neil Kelleher’s lab to develop the top down approach on a large scale, and after a decade of toil and trouble this has recently paid off.

His lab has been working on developing all three parts of the analysis process and, most recently, it has been the development of a more robust sample processing procedure, or a more robust way of fractionating intact proteins in solution, that has led to this recent breakthrough.

They have achieved a significant increase in scale with this improved sample processing, and can now analyse thousands of different protein isoforms within about a month. “This is a 20- to 30-fold increase in throughput, which is a major change,” says Kelleher.

“We can now analyse thousands of different protein forms and capture them from a single sample. This is still four times longer than the bottom up approach, but it is a major change in scale.

“And we are even able to get this to work on membrane proteins, which are very hydrophobic and represent a very difficult-to-analyse class of proteins that are of super-high interest to the pharmaceutical industry.”

Many of the proteins associated with the cell or plasma membrane are made up of hydrophobic amino acids that enable them to exist within the lipid bilayer of the cell membrane and bind tightly to it.

Extracting these hydrophobic proteins from the cell membrane has proven challenging in the past; they are difficult to solubilise, particularly in an intact form. And even when this is achieved, separating and extracting them from polyacrylamide gels for further analysis is then required.

Essentially, Kelleher’s team is characterising endogenous proteins – naturally occurring proteins that are not artificially digested by proteases.

There can be multiple post-translational modifications made on the same protein and the high-resolution flavour of mass spectrometry, along with special software that Kelleher’s team has developed, enables these modifications to be seen in automated mode.

Read part III of top down proteomics.