QPrEST+ - validated protein standards for targeted proteomics
QPrEST™ standards are stable isotope-labeled protein standards for the absolute quantification of human proteins using mass spectrometry. The premium QPrEST+ standards offer additional benefits including validation according to the CPTAC assay criteria.
QPrEST and QPrEST+
QPrEST protein standards consist of a heavy labeled 50-150 amino acid stretch identical to a part of the corresponding human protein. They are designed to have low sequence similarity to other proteins and theoretically form several tryptic peptides. QPrEST standards are accurately pre-quantified by LC-MS/ MS using an amino acid analyzed reference protein.
QPrEST standards are added to the sample of interest before the sample preparation and trypsin digestion. Due to identical amino acids surrounding the cleavage sites, peptides are subsequently formed and released similarly for both the QPrEST and the endogenous protein.
QPrEST standards are available for the majority of all human proteins and are generally produced on demand.
Premium benefits of QPrEST+
QPrEST+ standards are QPrEST standards with a minimum of one peptide validated according to the CPTAC assay criteria. QPrEST+ standards provide quantotypic peptides comparable to stable isotope-labeled full-length proteins. Furthermore, QPrEST+ standards show good linear responses for quantotypic peptides and are suitable for single-point calibration. A range of QPrEST+ standards are available on the shelf for selected human proteins.
Benefits of using QPrEST standards
- Sequence covers at least two theoretical tryptic peptides
- Accurately quantified using an amino acid determined reference protein
- Shipped in lyophilized form
- Digested together with the endogenous protein
Premium benefits of QPrEST+
In addition to QPrEST benefits, the QPrEST+ standards also offer:
- Validation of internal peptide(s) according to CPTAC
- Accuracy verified through cross-validation or orthogonal method
- Recommended spike-in level for single-point calibration in specified matrix
QPrEST+ peptide validation
QPrEST+ standards contain a minimum of one peptide validated according to the CPTAC assay criteria.
To identify the linear range (define slope and intercept), the limit of detection (LOD) and the lower limit of quantification (LLOQ), samples are prepared in the relevant matrix in a multipoint response reversed curve (at least six points).
The suitability of QPrEST+ peptides as quantotypic peptides is further validated by cross-validation by another peptide.
An alternative strategy, when cross-validation is not applicable, is to apply an orthogonal method for validation of the accuracy, e.g. an immunoassay or comparing the generated result to a well-defined reference value.
QPrEST+ products are validated in at least one sample type, e.g. human plasma or a tissue lysate, and are supplied with a recommended spike-in level to use as a starting point.
Learn about details and criteria for validation of QPrEST+ standards in our white paper "Validated Isotope-Labeled Protein Standard for Absolute Quantification Using Mass Spectrometry".
In order for a peptide to be suitable for quantification, it is important that it ionizes well and gives a good signal. It must also be proteotypic, meaning that the peptide sequence is unique to the protein of interest. In addition, it must be established that the peptide could function as a quantotypic peptide.
The definition of a quantotypic peptide depends on which type of stable isotope-labeled standard is used.
For QPrEST+, the following quantotypic definition is used:
• Formed during protease digestion
• Unique sequence for the protein of interest
• Cross-validated by another peptide or method
For QPrEST-products, even miscleaved peptides can be defined as quantotypic.
Single point calibration
QPrEST+ standards are well suited for use in single-point calibration experiments since they are pre-quantified with high accuracy and contain one or more validated peptide(s).
Furthermore, QPrEST+ standards are provided with a recommended spike-in levels that give labeled:unlabeled ratios close to one in a standard sample. To achieve good accuracy and repeatability for single point calibration a ratio close to one is preferred. However, since QPrEST+ peptides have a validated linear response, they generally provide good quantitative results also when measuring at rather large off-ratios (as long as both the heavy and light signals are not too close to the lower limit of quantification).