Absolute Protein Quantification

For example, studies on cellular protein complexes and their subunit stoichiometry require absolute concentrations of all complex-forming protein components and the stoichiometry of a complex can, therefore, not be extracted from relative quantitative data. Further, mathematical modeling to simulate and predict the behavior of biological processes, an essential component of systems biology studies, is dependent on the absolute protein concentrations to determine, for example, rate constants or kinetic fluxes. A third important advantage of absolute data is its suitability for comprehensive comparisons across datasets. Therefore, inter-organism, inter-laboratory or inter-proteomic platform comparisons become feasible. In addition, discovery and development of new therapeutic and diagnostic protein biomarkers benefit from absolute protein quantification. Finally, precise and accurate absolute protein quantification is of interest to the biomedical and  pharmaceutical industry, for instance, in areas such as therapeutic proteins, food safety, food allergies or sports drug testing. In summary, a wide range of important applications and benefits arise from the absolute measurements of protein abundances compared to relative quantitative values, justifying the usually more time-, cost- and labor-intensive workflows required.

The most precise and most frequently used proteomic approach for absolute protein quantification relies on the usage of calibrated isotope-labeled peptide or protein standards, which can be spiked into the sample(s) of interested at a precisely known concentration (for more detailed information please use the navigation menu on the left side of this page). While having the advantages of providing highly precise and potentially accurate absolute data, the spike-in approach is very cost intensive and can typically be applied to just a few analytes of interest. An alternative to isotope-labeled absolute quantification is absolute label-free abundance estimation, which is significantly less accurate (average error in the range of 2-fold), but significantly cheaper and applicable on a proteome-wide scale. BayBioMS offers label-based as well as label-free absolute protein quantification analyses, the optimal approach must get selected according to the specific biological question asked.

Outcome

The final outcome of an absolute quantitative proteomic analysis is a list of identified proteins or PTM-peptides. Further, for each protein and peptide an absolute protein concentration value is reported. The unit of this absolute value can be “protein copies per cell” or “picomoles of protein per volume of body fluid” or “protein weight per tissue or cell extract weight”, depending on which sample parameters have been previously investigate or are known for the underlying sample type. For example, for the determination of “protein copies per cell” the total number of cells that were used to extract a certain amount of proteins must have been determined, or the average protein amount per cell volume must be known.