General QPrEST Protocol

QPrEST standards are used for the determination of absolute protein concentrations in biological samples.

The sample of interest is spiked with QPrEST standard, digested and analyzed by mass spectrometry (MS). Signals from endogenous peptides are compared to those of QPrEST peptides and the resulting light to heavy (L/H) ratios are used to determine the absolute quantity of the endogenous protein in the sample. The QPrEST standards are easily incorporated into existing sample preparation workflows. Some general advice on handling the QPrESTs and suggested step by step protocols are presented below.

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QPrEST Handling

Reconstitution and storage

QPrEST standards are delivered lyophilized, from a buffer of 0.1 M Tris-HCl with 5 % Trehalose, in two vials with at least 0.5 nmol in each vial. Store the lyophilized QPrEST at -20°C. Spin the vial prior to opening to ensure that the standard is localized in the bottom of the vial. Reconstitute the QPrEST standard to the stated concentration by adding exactly 100 µL ultrapure water to each vial followed by thorough vortexing and spin down. The reconstituted QPrEST can be stored in aliquots at -20°C for four weeks. The thawed standard should be vortexed thoroughly and spun down prior to use.

Dilution

Depending on the target protein concentration in the sample of interest, dilution of the standard may be necessary prior to spike-in. In this case, it is recommended to make the dilutions in low protein binding tubes and preferably in a buffer at pH 8.0 containing 7-10 % sodium deoxycholate (SDC) to minimize dilution errors.

Sample Preparation

General

Samples spiked with QPrEST standards can be prepared using several different protocols, such as in-solution digestion or filter-aided sample preparation (FASP). When using the FASP method, care should be taken when choosing the filter unit pore size since the molecular weight of a QPrEST standard is approximately 25 kDa. The QPrESTs should be added as early as possible during the sample preparation and must be added to the samples before digestion. However, to ensure correct quantification, QPrEST should not be added to the samples before steps such as protein fractionation, enrichment or depletion where the QPrEST standard and the endogenous protein might end up in different fractions or where the ratio between them can be affected.

Spike in

When performing the QPrEST spike-in, it is advisable to use low retention pipette tips for maximal accuracy and precision of the analysis. Protein quantification can be achieved with a standard curve setup or by using a single spike-in level to obtain a one-point calibration.

When using a single spike-in level the QPrESTs should, if possible, be spiked in at an L/H-ratio close to 1. If a predicted concentration of the target protein is not available, it is advisable to perform an initial test experiment to determine the optimal QPrEST spike-in level. That type of experiment would include preparation and MS analysis of samples with QPrEST spike-in at three different concentrations in dilution steps of ten. For example, when analyzing plasma or cell lysate, the addition of 10, 100 and 1000 fmol QPrEST per µL plasma or per million cells are good starting points.  The test should reveal an approximate target concentration and a second experiment with an adjusted spike-in level can be performed.

Digestion*

Prior to digestion, denaturation, reduction, and alkylation are recommended. QPrEST standards contain heavy isotope-labeled arginine and lysine residues, why a proteolytic enzyme that cleaves specifically at these residues is preferred. Trypsin is recommended as it cleaves after both residues. Other enzymes specific for either of these sites, such as LysC or ArgC, may also be used or alternatively a combination of different enzymes (for example trypsin and LysC). A recommended trypsin total protein ratio is 1:50, note that plasma contains about 60 µg protein/µL and one million HeLa cells approximately 300 µg of protein.

* Also see suggested step-by-step instructions below. 

LC-MS Analysis

Digested samples spiked with QPrESTs can be analyzed with many different LC-MS instrument setups and acquisition modes e.g. multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data-independent acquisition (DIA) and data-dependent acquisition (DDA).

Data Analysis

Most proteomics data analysis software provides the functionality to easily export ratios between light and heavy peptides, from which the endogenous protein concentration can be determined. Since QPrEST standards can cover multiple peptides, several L/H ratios for the target protein will possibly be generated. The protein concentration can either be determined directly from the L/H ratios (single-point calibration) or by comparison to standard curves.

In general, the digestion efficiency is very similar between the endogenous protein and QPrEST standard. This means that even peptides containing missed cleavage sites in most cases show similar L/H ratios as fully tryptic peptides and therefore can also be used for quantification.

Occasionally separate peptides from the same QPrEST show different L/H ratios. There are several possible explanations for this such as post-translational modifications being present on one of the endogenous peptides, different protein isoforms present in the sample or the presence of natural sequence variants.

Guidelines for creating a targeted quantitative assay can be found at the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay Portal (https://proteomics.cancer.gov/assay-portal).

Digestion Protocols

Examples of step by step protocols for digestion of different protein sample types are described below. Prior to MS analysis, peptide desalting can be performed. A number of different products from different suppliers are available for this purpose, alternatively, desalting tips with C18 matrix can be generated in-house.

Digestion of cell/tissue lysates

This protocol for sample preparation of cell and tissue lysates are adapted from Geyer at al.1. In this protocol the cell lysis is performed in sodium deoxycholate (SDC), however, if another cell lysis or tissue preparation protocol including detergents not compatible with the described digestion protocol is preferred, filter aided sample preparation (FASP) procedure described by Wisniewski et al.2 could be used.

Procedure

Cell lysis

  1. Dilute cells or homogenates by adding four volumes of lysis buffer (0.1 M Tris-HCL or 25 mM NH4HCO3, 1% SDC, protease inhibitor, pH 8.0)
  2. Heat samples at 96°C for 10 min
  3. Use a 5 mL syringe with 23G needle to aspirate and dispense gently 10 times
  4. Homogenize the sample by sonication or equivalent treatment
  5. Centrifuge at 18,000 x g at 4°C for 30min
  6. Collect the protein supernatant

Digestion

  1. Add the desired amount of QPrEST standard to the protein sample. If a dilution of the QPrEST standard is needed prior to addition, dilute the QPrEST in 0.1 M Tris-HCl containing SDC to a final concentration of 7-10%.
  2. Add dithiothreitol (DTT) to a final concentration of 10 mM, incubate at 95°C for 10 min
  3. Allow tubes to cool down
  4. Add iodoacetamide (IAA) to a final concentration of 50 mM, incubate in dark at room temperature for 30 min
  5. Add dilution buffer (25 mM NH4HCO3 or 0.1 M Tris-HCl pH 8.0) to a final concentration of maximum 1% SDC
  6. Add trypsin (1:50 enzyme to substrate ratio) and incubate at 37°C overnight
  7. Quench digestion with 1% trifluoroacetic acid (TFA) to a final concentration of 0.5% and incubate at room temperature for 30 min
  8. Centrifuge at 13,000 x g for 10 min and transfer the supernatant to a new tube
  9. Perform desalting if needed

Digestion of plasma/serum

Plasma and serum samples can be prepared using in-solution digestion. The following protocol includes the ionic detergent sodium deoxycholate (SDC) and is adapted from Kuzyk et al.3.

Procedure

  1. Dilute plasma/serum 1:10 in dilution buffer (25 mM NH4HCO3 or 0.1 M Tris-HCl pH 8.0)
  2. Dilute the QPrEST to the desired concentration in dilution buffer containing SDC to a final concentration of 7-10%.
  3. Add the desired amount of QPrEST to the sample (adjust volumes needed for the MS analysis)
  4. If the QPrEST is not diluted add SDC the sample to a final concentration of 1-5%
  5. Add dithiothreitol (DTT) to a final concentration of 10 mM, incubate at 95°C for 10 min
  6. Allow tubes to cool down
  7. Add iodoacetamide (IAA) to a final concentration of 50 mM, incubate at room temperature for 30 min
  8. Add dilution buffer (25 mM NH4HCO3 or 0.1 M Tris-HCl pH 8.0) to a final concentration of maximum 1% SDC
  9. Add trypsin (1:50 enzyme to substrate ratio) and incubate at 37°C overnight
  10. Quench digestion with 1% trifluoroacetic acid to a final concentration of 0.5% and incubate at room temperature for 30 min
  11. Centrifuge at 13,000 x g for 10 min and transfer supernatant to a new tube
  12. Perform desalting if needed

References

1.Geyer, P.E, Kulak, N.A., Pichler, G., Holdt, L.M., Teupser, D., and Mann, M. Plasma Proteome Profiling to Assess Human Health and Disease, Cell Systems 3, 185-195 (2016).

2.Wisniewski, J.R., Zougman, A., Nagaraj, N. and Mann, M. Universal sample preparation method for proteome analysis. Nat Methods 6, 359-362 (2009).

3.Kuzyk, M.A. et al. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma. Mol Cell Proteomics 8, 1860-1877 (2009).

 

 

Rev. E. 2019 09 09