Protein quantification with mass spectrometry
3 May 2019
The development of analytical techniques has enabled applying mass spectrometry methods also for protein quantification from biological samples. The use of LC/MS brings in more analytical potential due to its versatility and accuracy and hence, its importance as an analytical tool for protein characterisation is increasing. If not being familiar with the topic, it may be interesting to have a peek how proteins are quantified from biological matrixes with LC/MS.
Targeted protein quantification means in practise monitoring the LC/MS signal of specific peptides in the sample. This is called “surrogate peptide approach” and for that, the sequence of the target protein has to be known. One of the critical steps in the quantification process is selecting the correct surrogate peptides. The selected peptides should be stable and represent the target protein in several locations of the sequence. This is a step, where bioinformatics tools are applied to choose unique peptide sequences that exist only in the target protein. One of the definite advantages of LC/MS analysis is that there is no absolute need to have a specific antibody and therefore, the method development is usually faster. However, antibodies may be used as capture reagents to increase sensitivity.
Another critical step is sample preparation. Prior the protein digestion, the sample should be optimised for the analysis of the target protein. This can be done either by enriching the target protein or depleting the background of the sample matrix proteome. Quantification of the target protein is done based on the concentrations of the enzymatically generated surrogate peptide and using standard proteins spiked at known concentrations and treated identically to the samples.
It must be noted that in order to produce reliable information, using the right software both in the selection of the surrogate peptides and data analysis, and having the state-of-the-art equipment for analysing the samples have a big role in the process. Naturally, on top of the right equipment, the scientists have to have the knowledge and experience how to approach the research questions and analytical challenges of the biologics to obtain high quality data. One might want to keep this analytical option in mind, when planning PK studies for biologics, as it may facilitate the progress of the project significantly. If you are not familiar with the approach, our experienced protein scientists are happy to help in setting up sensitive and accurate methods for quantifying proteins in biological matrixes!
Written by Miia Kovalainen