QPrEST - High Accuracy Targeted Proteomics

QPrEST™ standards are stable isotope-labeled standards for absolute quantification using mass spectrometry. In our catalog, you can find QPrEST standards targeting the majority of the proteins in the human proteome.

 

Unique Features of QPrESTs

QPrEST targeted proteomics logo

  • Designed to cover the most unique sequence within each human protein
  • Covers regions with several intrinsic proteotypic peptides
  • Added early on in the workflow
  • Delivers high accuracy quantification for a wide range of sample types

Each QPrEST standard consists of a heavy labeled 50-150 amino acid stretch 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 with multiple proteotypic peptides.

Benefits of the QPrEST Technology

QPrESTs are added to the sample early on in the workflow, prior to proteolytic digestion. The early spike-in and shared amino acid sequence of the QPrEST and the endogenous protein brings three major benefits:

  • Minimizing variation introduced during sample digestion
  • Similar digestion efficiency, enabling the use of miscleaved peptides, generating the same heavy to light ratios as fully cleaved peptides
  • Multiple intrinsic proteotypic peptides enabling cross-validation between peptides for quantification
ematic view over the workflow for protein quantification using QPrEST standards.
Schematic view over the workflow for protein quantification using QPrEST standards. The labeled QPrEST is spiked into the protein sample prior to trypsin digestion. The formed labeled and unlabeled peptides are then compared to determine the protein concentration.
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Interested in learning more about the unique QPrEST Technology? Download our white paper: QPrEST High Accuracy Targeted Proteomics

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QPrESTs are Pre-Quantified and Ready-to-Use

The N-terminal part of the QPrEST consists of a QTag sequence containing quantotypic peptides used for accurate quantification of the QPrEST (see image below). An unlabeled, ultra-pure and amino acid analyzed QTag, is used as an internal reference in an LC-MS-QTOF setup where ratios between the light and heavy QTag peptides are used to determine the absolute QPrEST concentrations5.

Schematic figure of a QPrEST standard.
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 using an unlabeled QTag. 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 the endogenous target protein.

Production and Quality Control

QPrESTs 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, Quality Control (QC) is performed after lyophilization. The QC includes the following steps:

  • QPrEST purity (≥90%), BioAnalyzer protein 230 purity assay
  • Protein identity (correct protein MW), LC-MS analysis
  • Presence of correct QPrEST peptides, LC-MS/MS
  • Isotopic incorporation (>99%), LC-MS

 

QPrESTs originate from the Human Protein Atlas project

QPrESTs originally stem from a research collaboration between Professor Mathias Uhlén and Professor Matthias Mann, who showed that the vast resource of Protein Epitope Signature Tags (PrESTs) produced within the Human Protein Atlas project also could be used as spike-in standards for mass spectrometry-based protein quantification in heavy labeled cell lines. For this purpose, a pipeline for the production of stable isotope labeled PrESTs was developed.

These stable isotope labeled PrESTs are now manufactured and quality controlled by Atlas Antibodies as QPrESTs, and soon commercially available for scientists worldwide.

Since the development of the QPrEST-based protein quantification method, the founding professors and several other groups have used the approach in their research, resulting in a number of publications. A selected number of publications are listed below in the References section.  

References

1. Aebersold R. and Mann M. (2003) Mass spectrometry-based proteomics. Nature 422, 198-207
2. Uhlén M. et al. (2010) Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 28, 1248-1250
3. Uhlén M. et al. (2015) Tissue-based map of the human proteome. Science 347, 6220
4. Edfors F. et al. (2014) Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Proteomics. 13,1611-24
5. 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
6. Zeiler M. et al. (2014) Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 13, 3435-3445,
7. Matic I. et al. (2011) Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples. J Proteome Res 10, 4869-4875
8. Edfors F. et al. (2016) Gene-specific correlation of RNA and protein levels in human cells and tissues. Mol Syst Biol. 12, 883