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Summary
Membrane proteins constitute approximately 30% of all cellular proteins. Nevertheless, this group of proteins is traditionally understudied due to limitations of the available analytical tools. Given the importance of membrane proteins in the various cellular processes and their potential as drug targets, NIST is developing methods for detection and quantification of membrane proteins by multiple reaction monitoring (MRM) mass spectrometry (MS).
Description
Membrane proteins constitute approximately 30% of all cellular proteins. Nevertheless, this group of proteins is traditionally understudied due to limitations of the available analytical tools. Given the importance of membrane proteins in the various cellular processes and their potential as drug targets, NIST is developing methods for detection and quantification of membrane proteins by multiple reaction monitoring (MRM) mass spectrometry (MS).
Major Accomplishments
- We have developed a universal workflow for sample processing and enrichment by optimizing washing and solubilization conditions and implementing sample fractionation by Whole Gel Eluter. The optimized sample preparation conditions and instrumental parameters allowed quantitative MRM measurements of membrane bound proteins at sub-pmol per mg tissue protein concentrations.
- We have applied this workflow to quantification of several clinically-relevant protein biomarkers and to analysis of extracellular vesicles.
Quantitative analysis of low-abundance membrane proteins requires further optimization of analytical methods developed for their soluble counterparts. Use of stable isotope-labeled full-length proteins as an internal standard prior to MRM analysis makes possible pre-fractionation of the target proteins and quantification of those low-abundance proteins, which cannot be reached without biological sample enrichment. This benefit can be used if a sample processing workflow allows entire solubilization of membrane proteins under conditions compatible with the following trypsinolysis and MRM analysis.