In this work, a triple quadrupole linear ion trap mass spectrometer has been utilized to create >1000 high quality, specific MRM transitions for multiple peptides to many plasma proteins. A non-isobaric chemical labelling strategy has been employed to create global reference standards to enable quantitative comparisons between clinical samples. This strategy has been applied to the study of 20 twin pairs, with the goal of understanding biological variation.

Plasma from 20 pairs of twins were collected and stored under identical conditions. Each sample was depleted (top 7 proteins), reduced/alkyated, and digested with trypsin. A global reference standard (GRS) was created from a pool of the digested patient samples by labelling it with the heavy mTRAQ reagent and added to each light labelled individual patient sample. By incorporating MRM transitions to this GRS in the assay, in addition to the MRMs to the sample, very high analytical reproducibility can be achieved for peptide ratios across measurements, typically 5% CV or better. MRM transitions (872) to 109 peptides for medium/high abundant plasma proteins were monitored. The protein ratios relative to the GRS were computed for all individual samples, providing an estimate of the biological variation across this small dataset. Proteins differed in the degree of variation, from low variation for proteins like Vitronectin and Clusterin, to higher variation in proteins like ApoA4, ApoH and Complement C4-A beta chain.

By comparing the differences between random individuals to the differences between members of a twin pair, the influence of genetics on the circulating levels of each protein can be estimated. When comparing the set of protein ratios, the intra-twin correlation was typically higher than the inter-twin correlation. Understanding the biological variation of specific proteins across populations is an important consideration in determining the utility of protein biomarkers.