QPrEST - High Accuracy Targeted Proteomics

QPrEST standards are stable isotope-labeled standards for absolute quantification using mass spectrometry targeting almost the entire human proteome.‚Äč QPrESTs are designed to target the most unique sequences of proteins, delivering high accuracy, reliable quantification. 

Each QPrEST standard consists of 50-150 amino acids identical to a part of the corresponding human protein. The sequences are carefully selected to have low similarity to other human proteins and cover regions wit h multiple proteotypic peptides.

Figure 1. Schematic figure of a QPrEST standard. The N-terminal part of the sequence consists of the QTag, used for purification and accurate quantification of the QPrEST. The C-terminal part of the sequence is identical to a portion of a human protein, this part is used by the end-user for absolute quantification of their target protein.

What are the benefits of QPrEST standards?

QPrEST targeted proteomics logo

One benefit of using QPrESTs for quantification of proteins is that they are added early in workflow, prior to proteolytic digestion, resulting in a decreased variation compared with using synthetic peptide standards which are often added after the digestion step.

The shared amino acid sequence of the QPrEST and the endogenous protein brings two additional major benefits:

  • Similar digestion efficiency, enabling the use of miscleaved peptides, generating similar heavy to light ratios as fully cleaved peptides
  • Multiple intrinsic proteotypic peptides enabling cross-validation between peptides for quantification

How do I use QPrEST standards?

Using QPrEST standards is easy. They come pre-quantified and ready to use and the simple workflow makes protein quantification straight forward. 

Figure 2. Schematic view of the workflow for protein quantification using QPrEST standards. The labeled QPrEST is spiked into the protein sample prior to trypsin digestion. The labeled and unlabeled peptides are then compared to determine the protein concentration.

How are QPrEST standards produced?

QPrEST standards show a near complete (>99%) isotopic incorporation. This is achieved through expression of the proteins in an auxotrophic Escherichia coli BL21(DE3) derivative with the addition of heavy isotope-labeled arginine and lysine (13C, 15N).

After cultivation, the proteins are affinity purified, using an N-terminal hexahistidine tag, and then lyophilized for long term storage.

To avoid any variation introduced during the lyophilization step, the following quality control steps are performed after lyophilization:

  • QPrEST purity (≥90%), Bioanalyzer protein 230 purity assay of intact protein
  • Protein integrity, LC-MS analysis of intact protein
  • Protein identity, LC-MS/MS analysis of digested protein
  • Isotopic incorporation (>99%), LC-MS/MS analysis of digested protein
  • Concentration determination, LC-MS/MS analysis of digested protein

References

The concept for QPrEST originated with Protein Epitope Signature Tags used in the Human Protein Atlas Project. Since then, many researchers have successfully utilized QPrESTs for protein quantification and published their findings.

  1. Al Shweiki et al. (2020) Proteomic analysis reveals a biosignature of decreased synaptic protein in cerebrospinal fluid of major depressive disorder. Translational Psychiatry, 10:144
  2. Hober, A et al. (2019). Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay. Mol. Cell. Proteomics 18, 2433–2446
  3. Edfors, F. et al. (2019) Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics Proteome Res 18, 2706−2718
  4. Valdés A et al. (2019) Development of a Parallel Reaction Monitoring-MS Method To Quantify IGF Proteins in Dogs and a Case of Nonislet Cell Tumor Hypoglycemia. J Proteome Res 18, 18-29
  5. Andersson A et al. (2019) Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease. Clin Chim Acta, 79-93
  6. van Steenoven I et al. (2019) VGF Peptides in Cerebrospinal Fluid of Patients with Dementia with Lewy Bodies. Int J Mol Sci 20, 4674
  7. Oeckl P et al. (2018) Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid. J Proteome Res 17, 516-523
  8. Edfors, F. et al. (2016) Gene-specific correlation of RNA and protein levels in human cells and tissues. Mol. Biol. 12, 883
  9. Edfors F. et al. (2014) Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Proteomics 13, 1611-24
  10. Zeiler M. et al. (2012) A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.Mol Cell Proteomics 11, O111 009613