Stable Isotope-Labeled Protein Standards for Absolute Quantification Using Mass Spectrometry

Mass spectrometry (MS) is considered the preferred technique for protein analysis (1) in the field of proteomics. Using MS, thousands of proteins from a trypsin digested complex sample can be identified in a single run, alternatively, provide an absolute quantification for selected targets. One strategy for accurate quantification is to spike in heavy isotope-labeled standards corresponding to the proteins of interest. 

QPrEST Stable Isotope-Labeled Standard

Design

The QPrESTs originally emerged from the Human Protein Atlas project (2, 3), in which unlabeled (light) protein epitope signature tags or PrESTs are utilized as antigens for producing antibodies.

These PrESTs, measuring 50–150 aa long,  are designed to cover the most unique sequence within every human protein and to include multiple potential proteotypic peptides.

The QPrESTs are essentially heavy isotope-labeled forms of the PrESTs, and their unique sequence design makes them perfect for MS-based quantification in complex samples (4–6).

Workflow

The sample is spiked with QPrESTs during the early phase of the workflow, that is, before proteolytic digestion. The early spike-in and the shared sequence between the QPrEST and the endogenous protein results in higher accuracy and reduced variation when compared to methods using synthetic peptide standards, as shown in Figure 1. 

Schematic view over the workflow for protein quantification using QPrEST standards.

Figure 1. Schematic view over the workflow for protein quantification using QPrEST standardsThe 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.

 

Within the QPrEST, the presence of endogenous cleavage sites, due to the correct endogenous sequence, also enables the use of peptides containing missed cleavage sites. This can be attributed to the highly analogous digestion efficiency that typically produces heavy to light ratios very similar to the fully cleaved peptides. In addition, because of the sequence length, trypsin digestion releases numerous peptides that can allow quantitative results from different peptides to validate one another.

Quantification Tag

An N-terminal quantification tag (QTag) is present in all QPrEST standards. This is a protein sequence in which inherent peptides are utilized to precisely quantify the QPrEST (see Figure 2).

Schematic figure of a QPrEST standard. The N-terminal part of the sequence consists of the QTag sequence, 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 for absolute quantification of the endogenous target protein.

Figure 2. Schematic figure of a QPrEST standard. The N-terminal part of the sequence consists of the QTag sequence, used for 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 for absolute quantification of the endogenous target protein.

Given that there are numerous QTag peptides and that they are suitably distributed over the LC gradient, they can also be used as standards for retention time calibration (iRT), as illustrated in Figure 3. Since the QTag peptides are produced during the QPrEST digestion, additional sample preparation steps are not required.

Example chromatogram showing the distribution of QTag peptides over a 30 min LC gradient.

Figure 3. Example chromatogram showing the distribution of QTag peptides over a 30 min LC gradient.

Production and Quality Control

Heavy isotope-labeled QPrESTs are expressed in an Escherichia coli BL21(DE3) derivative — auxotrophic for arginine and lysine (7). Cells are cultivated in a minimal autoinduction medium and isotopic incorporation is realized by adding heavy isotope-labeled lysine and arginine (15N, 13C).

An N-terminal hexahistidine tag (part of the QTag) is used for the affinity purification of the QPrESTs, after which they are lyophilized for maximum stability and to facilitate storage for a long time.

Post reconstitution, the BioAnalyzer protein 230 purity assay is used to verify the QPrEST purity (≥90%), and LC-MS analysis is performed to confirm the exact protein molecular weight. For additional confirmation of the protein identity, LC-MS/MS is used to confirm the presence of correct QPrEST peptides produced by tryptic digestion.

The isotopic incorporation for QPrESTs is almost complete (>99%), which is confirmed by the lack of peaks corresponding to unlabeled peptides in an MS spectrum (see Figure 4).

Verification of isotopic incorporation. Analysis of QPrEST tryptic digests using ESI-MS shows that no peaks corresponding to unlabeled peptides can be detected.

Figure 4. Verification of isotopic incorporation. Analysis of QPrEST tryptic digests using ESI-MS shows that no peaks corresponding to unlabeled peptides can be detected.

Quantification

Each QPrEST is pre-quantified at Atlas Antibodies in a two-step method. First, an amino acid analysis of a highly pure (>98%) internal standard (the unlabeled QTag). This is followed by an LC-MS-QTOF setup, in which ratios between the heavy and light QTag peptides are used for establishing the absolute concentration of QPrEST (5). See Figure 2 above.  

To guarantee the QPrESTs’ precision and accuracy (CV ≤ 10%), the concentration is established based on the preparation and analysis of three replicates at three separate occasions.

Accuracy and Digestion

In order to assess the digestion efficiency of QPrEST standards in comparison to endogenous proteins, heavy to light peptide ratios produced after different time points of digestion were assessed. After cell lysis, QPrEST standards were directly added to HeLa lysates and the amount added was such that the generated heavy to light ratio was approximately 1:1. Subsequently, digestion was ceased at eight different time points ranging from 5 minutes to 16 hours.

Using data-dependent acquisition, samples were examined on a Bruker Impact II mass spectrometer. Heavy to light ratios, for a minimum of two peptides from each QPrEST, were established and plotted against digestion time.

Results shown in Figure 5 A and B demonstrate highly analogous digestion efficiency between endogenous peptides and QPrEST standards when permitting the proteolytic digestion reaction to reach equilibrium.

Heavy to light (H/L) ratios for peptides originating from GAPDH (A) and HSPD1 (B) after different digestion times. QPrEST sequences are shown at the bottom and quantified peptides are highlighted.

Heavy to light (H/L) ratios for peptides originating from GAPDH (A) and HSPD1 (B) after different digestion times. QPrEST sequences are shown at the bottom and quantified peptides are highlighted.

Figure 5. Heavy to light (H/L) ratios for peptides originating from GAPDH (A) and HSPD1 (B) after different digestion times. QPrEST sequences are shown at the bottom and quantified peptides are highlighted.

Data from the different peptides targeting the same protein also revealed similar outcomes (for fully cleaved peptides and missed cleaved peptides), thus confirming the quantification accuracy.

Application Examples

In complex samples, proteins can be quantified in a multiplex or a singleplex format using QPrEST standards for the proteins of interest (4, 5).

Protein Quantification in Cell Lines

Figure 6 shows the results of protein quantification using QPrESTs, wherein a set of QPrEST standards was spiked into a HeLa cell lysate, followed by digestion of the sample with trypsin utilizing the filter-aided sample preparation (FASP) methodology (4).

Absolute quantification of two human proteins using QPrEST standards. The copy numbers of CAPG and UGDH were determined in HeLa cells.

Figure 6. Absolute quantification of two human proteins using QPrEST standards. The copy numbers of CAPG and UGDH were determined in HeLa cells.

Following sample preparation, samples were examined on a Q Exactive HF mass spectrometer with the help of a data-dependent acquisition setup. The protein UGDH was measured to 890,000 copies for each cell using a total of five peptides produced from two independent QPrEST standards.

With regards to CAPG, a single QPrEST with a total of four peptides was utilized for quantification and the result demonstrated that this protein is present in HeLa cells at 2.5 million copies for every cell.

Protein Quantification in Plasma

In addition, QPrEST standards have been used for producing targeted assays in human plasma samples, either using a serial dilution method or a single-point-spike-in (see Figure 7a and b).

A QPrEST targeting APOA4 was serially diluted and spiked into plasma samples to establish the range for which the assay provides a linear response.

Using an in-solution sodium deoxycholate (SDC) protocol, digestion was carried out with trypsin and subsequently examined on a Q Exactive Plus mass spectrometer using a PRM targeted MS acquisition method.

Skyline software was used to process the PRM data and the extracted heavy to light ratios were subsequently plotted against the spiked-in QPrEST concentrations. Se Figure 7 A.

Absolute concentrations of APOA4 in plasma samples were determined at different QPrEST spike-in levels (A).

Figure 7A. Absolute concentrations of APOA4 in plasma samples were determined at different QPrEST spike-in levels. 

In total, both light and heavy peptides were identified for four peptides at nine QPrEST spike-in levels, with the response curve demonstrating excellent linearity (R2 = 0.994) across almost three orders of magnitude for all the peptides included.

On the basis of this data, it can be concluded that consistent quantitative results for APOA4 are achieved when the ratio of endogenous QPrEST spike-in levels and APOA4 is between 1:20 and 20:1.

Figure 7B shows the quantification of APOA4 in triplicate using a single spike-in level close to the endogenous APOA4 level. All four peptides exhibited analogous results, and based on the median of the four peptides, the APOA4 concentration in this plasma sample was found to be 2.6 µM. See Figure 7B.

Absolute concentrations of APOA4 in plasma were determined using a spike-in level close to the endogenous APOA4 concentration (B).

Figure 7B. Absolute concentrations of APOA4 in plasma were determined using a spike-in level close to the endogenous APOA4 concentration (B).

 

QPrEST Standards in Published Research

Professors Mathias Uhlén and Matthias Mann originally developed the QPrEST-based protein quantification technique, and subsequently, both of them have applied the method in their respective laboratories, leading to several publications.

In 2012, Zeiler et al. (5) demonstrated how QPrEST standards (then known as heavy isotope–labeled PrESTs or SILAC-PrESTs) could be employed for multiplex protein quantification in HeLa cells. In that proof-of-principle study, complete quantification of 40 human proteins was carried out simultaneously. The included proteins covered a wide concentration range — from FOS with a cellular abundance of 6,000 copies, to vimentin with 20 million copies for each cell.

In the year 2016, Edfors et al. (8) successfully quantified 55 proteins across 11 tissues and 9 cell lines using a multiplex QPrEST mix. This was done to study the per-gene correlation of mRNA and protein levels in various samples. In this study, the team also applied QPrESTs-based histone normalization approach to enable the determination of copy numbers for each cell in tissues with different cell densities.

Recently, Oeckl et al. (9) performed a comparison of different MS standards as alternatives to stable isotope-labeled proteins (PSAQ). The comparison included winged SIL (WiSIL) peptides, heavy peptides, and QPrEST standards, which were used for quantifying alpha-synuclein in cerebrospinal fluid, or CSF, through a selected reaction monitoring (SRM) MS setup. All methods produced results with satisfactory CV values (<15%), whereas QPrEST was the only standard that produced a concentration similar to the PSAQ method (deviation <15%).

Summary

  • QPrESTs are Stable Isotope-Labeled Protein Standards for Absolute Quantification using Mass Spectrometry
  • QPrESTs are spiked in early and show a similar digestion efficiency to the endogenous proteins, thus increasing accuracy in quantification
  • QPrESTs can be used to determine absolute protein amounts in multiple sample types, including cell lysate and plasma
  • QPrESTs are pre-quantified, “Ready to Use” and available for the vast majority of the proteins in the human proteome

References

  1. Aebersold R. and Mann M. (2003) Mass spectrometry-based proteomics. Nature 422, 198-207.
  2. Uhlen 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.
  4. Edfors F. et al, (2014) Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Prot. 13, 1611-1624.
  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., Danielsson, F., Hallström, B. M., Käll, L., Lundberg, E., Pontén, F., Forsström, B., Uhlén, M. (2016) Gene-specific correlation of RNA and protein levels in human cells and tissues. Mol Syst Biol 12, 883.
  9. Oeckl, P., Steinacker, P., Otto, M., (2018) Comparison of internal standard approaches for SRM analysis of alpha-synuclein in cerebrospinal fluid. J Proteome Res 17, 516-523.

About Atlas Antibodies

 

The QPrEST products will be available later this year from Atlas Antibodies.  

Building on the heritage from the Human Protein Atlas project, Atlas Antibodies provide highly-validated reagents that enable leading research in biology, diagnostics, and medicine for the understanding and improvement of human health

 


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Last updated: Jun 11, 2019 at 4:10 AM

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