Protein quantification strategies based on isotopic dilution of Stable Isotope Standard (SIS) peptides or recombinant proteins provide precise and robust read-out across different analytical conditions. The introduction of internal standards as the first step in proteomics sample preparation workflows results in several advantages, including the ability to control for technical bias originating from enzymatic digestion of proteins into peptides. Here, we describe a multiplex protocol for targeted proteomics based on vacuum-dried recombinant protein standards, which enable a robust workflow with low technical variance (CV < 10%) between runs. Based on this novel concept, it is possible to store SIS proteins at room temperature, without compromising the quantitative performance and reproducibility of the analysis, allowing for a flexible protocol simply adding plasma or serum directly to the vacuum-dried mixture. Here, a set of 292 peptides towards 100 protein targets, spanning four orders of magnitude, were assayed in plasma with a median inter-assay variation of 4.6%. The targeted proteomics protocol was further improved by re-design of the protein standards to select proteotypic peptides with rapid and reproducible kinetics during the proteolytic digestion step. In summary, a novel concept for targeted proteomics is described, suitable for precision medicine efforts and high-throughput clinical analysis.

Here, we describe a novel approach in which many different isotope-labeled protein fragments are mixed in ratios representing the blood concentrations of the corresponding target proteins. The mixture is dried down into a salt pellet to decrease the assay volume and to facilitate storage. We show that this vacuum-dried pellet can be stored at room temperature for at least one month and be subsequently used for targeted proteomics analysis. The effect of repeated freeze-thaw cycles shows that the majority of the recombinant protein standards stored in this salt pellet are stable (CV<10 %) over repeated freeze-thaw cycles, and for at least six months. The targeted proteomics analysis is performed by simply dissolving the pellet with patient plasma followed by a standard protocol involving enzyme digestion and analysis using mass spectrometry. The concentration of the target proteins in the plasma sample is thus determined by comparing the ratio between peptides originating from the isotope (heavy) standard and the plasma-derived (light) sample.

Human blood plasma, collected in EDTA-tubes.