Sophia Doll (Presenter)
Max Planck Institute for biochemistry
Authorship: Sophia Doll1*, Philipp Geyer1*, Scarlet Beck1*, Lesca Holdt2, Daniel Teupser2, Matthias Mann1*
(1)Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany (*) NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark (2) Institute of Laboratory Medicine, Ludwig-Maximilians University Munich, 8
| Short Abstract We evaluated the use of Protein Epitopes Signature Tags (PrESTs) (Zeiler et al., 2012) for mass spectrometry-based absolute quantification of clinically relevant proteins in plasma samples. We showed that single or mixtures of PrESTs can be spiked into plasma for high accurat quantification of clinically relevant proteins, such as C-reactive protein (CRP). We think that such applications can be translated into the clinics for high-throughput quantification of biomarkers. |
Long Abstract
Human plasma is not only the most collected sample but also one of the richest versions of the human proteome since blood communicates with almost all cells that release part of their content into the bloodstream. The identification and quantification of the plasma proteome by mass spectrometry (MS)-based proteomics remains challenging due to the vast dynamic range of protein abundances. Only a handful of very abundant proteins represent 99% of the total protein content and mask the identification of low abundant proteins, which are of particular interest for the identification of biomarkers. While antibody-based depletion of abundant proteins enables the identification of up to 5,000 plasma proteins, using such technology leads to bias because of antibody cross-reaction or the elimination of clinically relevant proteins that bind to high abundant proteins, such as albumin. Recently, a rapid, high-throughput and highly reproducible workflow has been developed (Geyer et al. 2016) that allows the detection and quantification of hundreds of plasma proteins from a fingerprick of blood.
In the clinic, however, quantification relies on absolute values, which are often generated with the use of internal standards. Mass spectrometry-based absolute quantification is more challenging than relative quantification and requires the addition of labeled internal standards. To add this capability to our automated workflow, we evaluated the use of so called SILAC-PrESTs, which are recombinant expressed stable isotope labeled protein fragments, used as internal plasma standards. This approach has the advantage (compared to AQUA peptides for example) to control for sample processing. The ratio of an SILAC internal standard to an endogenous peptide enables the absolute quantification of the protein of interest.
We showed that single, but also mixtures of several clinically relevant plasma proteins can be quantified with high accuracy using PrESTs. For example, C-reactive protein (CRP) levels were quantifiedand by using a master mix of four apolipoproteinsproteins (APOA1, APOA4, APOB, APOE) we showed that these proteins can be quantified with high reproducibility in human plasma samples. Indeed, our data showed low intra-assay CVs (APOA1 = 2.3%, APOA4 = 3.8%, APOB = 5.3%, APOE = 3.8%).
To improve the detection of PrESTs, we tested different MS measurement methods using different resolutions as well as scanning modes. We also showed that no cross contamination of PrESTs was observed during sample preparation and we optimized the short measurement gradients to reduce sample carry over one liquid chromatography run to another.Finally, we showed that PrESTs facilitate the detection of low abundant plasma proteins.
In summary, we developed a rapid and high-throughput workflow, which allows the absolute quantification of multiple proteins and which could be transfer into future clinical application.
References & Acknowledgements:
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