Feature: Tackling proteomics from the top down

By Susan Williamson

Conceptually, it’s a relatively straightforward task, but providing a high fidelity inventory of protein molecules that are naturally present inside human cells or tissues is not quite as simple as it sounds.

The problem is, the proteins – wily things that they are – don’t remain static. Post-translational modifications make the protein inventory a tricky thing to pin down, particularly when basing your study on the underlying gene sequence.

For over a decade, Professor Neil Kelleher, and his team at the University of Illinois, has been developing technology to tackle this so-called ‘isoform problem.’ And the approach he’s been developing might just change the way in which we work towards establishing the full catalogue of human proteins.

“This work is producing a major shift in how proteins can be analysed by mass spectrometry,” says Kelleher. “I see this major shift can and will happen during this decade.”

The so-called ‘top down’ strategy is at the core of Kelleher’s work. This approach involves measuring whole proteins, and enables the fundamental molecular biology at a molecular level to be understood.

Most proteome analysis is currently done the other way around: the ‘bottom up’ approach, where a complex sample of proteins is digested with proteases and then analysed.

The benefits of the bottom up approach are that thousands of proteins can be detected in a single run. The problems is that it involves a lot of technical processing and, because it is a peptide-based strategy, the different protein isoforms cannot be detected and characterised with precision.

“The average protein is digested into 40 peptides and this creates lots of work to piece these bits back together to decipher the whole protein,” explains Kelleher. “Conceptually, the top down approach is easier than the bottom up.”

Kelleher believes that the unambiguous characterisation of entire protein sequences and their post-translational modifications are often best achieved through the analysis of intact proteins. Genes can be sequenced to decipher these different protein products; however, gene sequencing does not reveal the post-translational modifications that create the different protein isoforms.

“Digesting proteins into peptides obscures this complexity as well,” said Kelleher, referring to the complexity post-translational modifications bring to a protein, “whereas the top down approach keeps this complexity intact and reads it out directly.”

Read part II of top down proteomics.

Read part III of top down proteomics.