Work carried out at the University of Manchester has demonstrated a simple way to measure the abundance of protein molecules more accurately, which could help improve the reliability of many areas of biological and clinical research.

Improving peptide linearity // Image by Adam Byron

Measuring the abundance of protein molecules is a key part of many fields of life science and medicine, from analysing blood tests to studying human development to discovering new drugs. For proteomics – the study of all the proteins in a given system – reliably quantifying many proteins is an important yet challenging goal.

Accurate measurement is tricky because proteins are relatively large molecules, so scientists must often count protein fragments called peptides instead. The trouble is, proteins and peptides can stick to the containers used to store them and to the equipment used to measure them.

Building on previous research, the new study shows that peptide stickiness greatly reduces the accuracy of peptide quantification.

Accurate quantification of proteins is a key capability.

By making over 2100 measurements using a mass spectrometer (which is actually not that many in modern-day proteomics standards, but which enabled us to describe our findings), we showed that the detection of many peptides did not increase linearly as the amount of protein increased. A straight line graph of peptide signal intensity against protein concentration means you can calculate an unknown protein concentration from the intensity (see image, above). But a nonlinear peptide relationship makes it virtually impossible to work out the identity and abundance of protein molecules in a sample.

To overcome this problem, we found that the addition of a small amount of organic solvent to peptide samples dramatically improved the reliability of peptide quantification. The solvent worked a bit like a lubricant, preventing the sticking of peptide molecules.

A simple solution to a sticky struggle.

Effect of organic solvent on accuracy of peptide quantification // Image by Adam Byron // Adapted from Warwood et al. (2013) Journal of Proteomics 85, 160-164

The work has been published as an open-access article in today’s issue of the Journal of Proteomics.

This method will be important for biologists studying cells of our body or model organisms, such as yeast, and for clinicians investigating drug targets or biomarkers of disease. For these fields and many others, accurate quantification of proteins is a key capability.

Funding: This work was supported by the Wellcome Trust.

Citation: S Warwood, A Byron, MJ Humphries, D Knight, The effect of peptide adsorption on signal linearity and a simple approach to improve reliability of quantification. J. Proteomics 85, 160–164 (2013). HTML | PDF | PubMed

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