Targeted Proteomics FAQ
Find answers to the most commonly asked questions regarding QPrEST products and technology.
Questions on QPrESTs
QPrESTs are Stable Isotope-Labeled Protein Standards for Absolute Quantification using Mass Spectrometry. QPrESTs contain a stretch of approximately 50 to 150 amino acids identical to a human protein sequence, covering regions with multiple intrinsic proteotypic peptides.
The QPrESTs are expressed in an Escherichia coli (E.coli) strain auxotrophic for Lysine and Arginine, BL21 (DE3) ∆lysA ∆argA. The cultivation is performed in a minimal auto-induction medium with heavy isotope-labeled versions of Lysine and Arginine, ensuring >99% isotopic incorporation.
The amino acids are L-Arginine*HCl (U-13C6, U-15N4) and L-Lysine*2HCl (U-13C6, U-15N2).
The QPrESTs are purified using immobilized metal ion affinity chromatography using a HisPur cobalt resin under denaturing conditions.
All QPrESTs contain an N-terminal QTag, used for accurate determination of the QPrEST concentration. The QTag sequence is:
The specific human protein sequence begins directly after “VDKLAAA”.
The unlabeled QTag has a sequence identical to the QTag sequence present within the QPrEST. This unlabeled QTag is used as an internal standard to correctly quantify each QPrEST. The unlabeled QTag has a high purity (≥98%) which is achieved by the combination of two different types of affinity chromatography using both an N-terminal and a C-terminal purification tag. The amino acid analysis is used to accurately determine the concentration of the unlabeled QTag.
The unlabeled QTag is used as an internal reference to determine the absolute concentration of the QPrEST. After digesting a mixture of QPrEST and QTag standard, the QTag peptide ratios determined in mass spectrometry analysis are used to determine the abundance of the QPrEST, resulting in an accurate and precise concentration.
The QPrEST concentration is determined using the unlabeled QTag. The experiment is performed in triplicates generated at three different occasions, resulting in a precision equal to or better than 10%. The determined concentration of the QPrEST and the standard deviation determined from the three replicates is stated on the product datasheet for each individual product.
IMAC purification enables a purity of at least 90%. However, because of the quantification strategy, using an internal standard (QTag), contaminating E.coli proteins will not affect the quantification of the QPrEST nor the target protein when using the QPrEST as a standard protein.
The incorporation efficiency of heavy isotope-labeled Arginine and Lysine is at least 99% and is verified by MS/MS analysis of a digested QPrEST sample.
Each QPrEST contains a stretch of approximately 50 to 150 amino acids identical to a human protein sequence. The fragment length in combination with the selection of the most unique stretches within the endogenous proteins ensures that each QPrEST covers multiple intrinsic proteotypic peptides. QPrESTs can contain up to ~10 peptides.
The QPrESTs are lyophilized to ensure long term stability of the protein. The product is shipped in two separate vials so that you can plan and execute your experiments over time.
The QPrESTs should be added to the sample of interest prior to enzymatic digestion. If fractionation or purification is performed on the protein level, the QPrEST should be added after this step.
Lyophilized QPrEST should be stored at -20°C for up to one year. Reconstituted QPrESTs should be stored at -20°C for up to four weeks.
Each vial of QPrEST should be reconstituted using 100µl of MilliQ grade H2O. If further dilution of the QPrEST is needed, the dilution should always be performed using a buffer consisting of 0.1 M Tris-HCl, 10% sodium deoxycholate (SDC), pH 8.0.
For optimal quantification of the protein, the QPrESTs should be spiked in to the sample of interest aiming to achieve an H/L ratio close to 1. Preferably, generated ratios should be between 0.1 and 10.
A theoretical tryptic QPrEST peptide is between 7 and 35 amino acids long and contains a single K or R residue, which is located at its final position. For the defined theoretical peptides, trypsin is assumed not to cleave at KP and RP sites.
The QPrESTs have been used in several research publications. Some of the references are listed below.
Aebersold R. and Mann M. (2003) Mass spectrometry-based proteomics. Nature 422, 198-207
Uhlen M. et al. (2010) Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 28, 1248-1250
Uhlén M. et al. (2015) Tissue-based map of the human proteome. Science 347, 6220
Edfors F. et al. (2014) Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Proteomics. 13,1611-24 DOI: 10.1074/mcp.M113.034140
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 DOI: 10.1074/mcp.O111.009613
Zeiler M. et al. (2014) Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 13, 3435-3445 DOI: 10.1074/mcp.M114.038513
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
Edfors et al. (2016) Gene-specific correlation of RNA and protein levels in human cells and tissues. Mol Syst Biol. 12, 883
Oeckl et al. (2018) Comparison of internal standard approaches for SRM analysis of alpha-synuclein in cerebrospinal fluid. J Proteome Res 17, 516-523