Protein Quantification Using Validated Stable Isotope-Labeled Standards

Proteins carry out a wide range of functions inside of the human body and because of this, they are regularely quantified in global research from early discovery to the clinic.

In mass spectrometry-based proteomics, various label-free quantification techniques have been employed to provide relative quantification. However, there is often a requirement to establish absolute concentrations, such as in clinical environments, where samples are evaluated in comparison to a normal reference interval.

The preferred technique for absolute protein quantification in targeted investigations is to utilize stable isotope-labeled spike-in standards along with external calibration curves.

Validated Isotope-Labeled Protein Quantification

Figure 1. Proteomic workflow: Stable isotope-labeled standards used as internal standards for protein quantification are spiked in at different stages of the sample preparation. The stable isotope-labeled peptide standards are added to the sample after digestion and only normalize for variation occurring thereafter (dark gray). Stable isotope labeled protein standards are spiked in early in the preparation, before the trypsin digestion and the peptides are formed simultaneously as for the protein to be quantified (light gray). Image Credit: Atlas Antibodies

Stable Isotope-Labeled Standard Approaches

The most frequently used stable isotope-labeled standards for the quantification of proteins are synthesized peptides that are introduced after the sample has been reduced, alkylated and digested (Figure 1).

These labeled peptides can normalize for variation in the later steps which comprise of sample injection, LC-MS/MS analysis, and peptide cleanup, but they cannot explain differences occurring throughout the process of digestion.

The stable isotope-labeled full-length proteins are a more dependable choice. These are introduced prior to the sample preparation and digestion, which creates a digestion efficiency akin to the quantified protein.

A limited number of stable isotope-labeled full-length proteins are available for purchase and they are normally costly, while labeled peptides are commonly available and are frequently synthesized on demand.

QPrEST

Quantitative Protein Epitope Signature Tags (QPrESTs) are stable isotope-labeled protein standards produced for absolute quantification.

The QPrEST sequence exactly matches a section of a human protein and is designed to have low sequence similarity to other proteins and theoretically produces multiple tryptic peptides. The QPrESTs are precisely pre-quantified by LC-MS/MS utilizing an amino acid analyzed reference protein.

As the stable isotope-labeled full-length proteins, the QPrEST standards are introduced to the sample of interest prior to trypsin digestion and sample preparation (Figure 2).

Peptides are then produced and released in the same way for both the endogenous protein and the QPrEST, this due to the length of the QPrEST and the identical amino acids surrounding all cleavage sites. The QPrESTs are produced on demand and cover the majority of the human proteome.

Suggested workflow for quantification using QPrEST: Accurately quantified lyophilized QPrEST is reconstituted and spiked into the sample prior reduction, alkylation, and trypsin digestion. After peptide cleanup, the sample is analyzed using LC-MS/MS and the data is analyzed with a quantification software using single-point calibration.

Figure 2. Suggested workflow for quantification using QPrEST: Accurately quantified lyophilized QPrEST is reconstituted and spiked into the sample prior to reduction, alkylation, and trypsin digestion. After peptide cleanup, the sample is analyzed using LC-MS/MS and the data is analyzed. Image Credit: Atlas Antibodies

Quantotypic Peptides

Multiple criteria must be attained for a peptide to be appropriate for quantification. A peptide must firstly ionize sufficiently to emit a strong detectable signal in the mass spectrometer, and secondly, it should be proteotypic, meaning it has a sequence exclusive to the protein of interest.

A proteotypic peptide that provides a strong signal must be further assessed to determine whether it could also perform as a quantotypic peptide. For example, amino acids and motifs of predicted or determined post translational modifications should be excluded in quantotypic peptides.

The definition of a quantotypic peptide is reliant upon the kind of stable isotope-labeled standard employed. For instance, for synthetic peptides, N-terminal glutamine residues should be avoided since these peptides can be subjected to cyclization which will produce pyroglutamate. Any changes like these, prior to the addition of the sample, will compromise the accuracy and precision of the quantification.

Proteins (QPrEST and Full Length) are generally more robust in solution when compared to peptides, however  modification sites in the quantotypic peptides should preferably be avoided even for these.

Modifications happening after the introduction of the protein standards to the sample are theoretically produced in the same way in both the protein of interest and the standard and would not impact the quantification accuracy.

Peptides with surrounding cleavage sites that comprise of several lysines or arginines are normally only partially digested, which means that these should be avoided for the peptide standards.

Stable isotope-labeled protein standards, such as QPrESTs, are more consistent. Using these standards even miscleaved peptides could be established as quantotypic peptides because the tryptic peptides are produced in the same way for the quantified protein and the protein standards.

The following is the quantotypic peptide definition for stable isotope-labeled QPrEST protein standard:

  • Produced at the moment of protease digestion
  • Exclusive sequence for protein of interest
  • Cross-validated by a further technique or peptide

Additionally:

  • Miscleaved peptides can also be employed for QPrEST products

CPTAC Assay

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a public repository of MS-based, well-characterized targeted proteomics assays. They are precise and specific, and can be reproduced and standardized in order to be supplied across laboratories.

An assay is defined as the quantification of a peptide in an intricate blend of peptides produced after the proteolytic digestion of an appropriate sample matrix.

The research community can share and upload data about an assay on the CPTAC portal, two studies are needed for upload:

  • Repeatability of the assay
  • Linearity of a response curve and lower limit of quantification

QPrEST+ Peptide Validation

QPrEST+ is a QPrEST protein standard containing at least one peptide that has been validated in line with the CPTAC assay criteria.

To establish the linear range (specify the intercept and slope), the lower limit of quantification (LLOQ), and the limit of detection (LOD), samples are prepared in the appropriate matrix in a multipoint response reversed curve (six points at minimum). The analysis includes three transitions for each peptide and their results should be within ±30%.

Three concentrations (low, medium and high) are determined to access the repeatability (intra- and inter-assay variability) of the assay, this is done using three replicates at five alternate occasions. Approved intra- and inter assay variation (CV%) must be less than 20%.

To further verify the suitability of QPrEST+ peptides as quantotypic peptides, techniques produced in-house are applied. As a first choice, the peptide result created can be cross-validated by an additional peptide.

It is a requirement that the generated ratios are verified (±20%) by at least one other QPrEST peptide (the peptide could derive from the same or a further QPrEST) in the same sample utilizing this technique.

If this is not applicable, other methods including the use of an orthogonal technique for concentration validation, such as immunoassay, or comparing to a global reference value can be performed.

The QPrEST+ products are verified in a select sample type, such as a tissue lysate or human plasma, and are supplied with a suggested spike-in level.

Advantages of using QPrESTs

  • Sequence covers multiple theoretical tryptic peptides
  • Precisely quantified by an amino acid determined reference protein
  • Delivered in lyophilized form
  • Digested along with the endogenous protein

Further benefits of QPrEST+

  • Internal peptide(s) are validated in line with CPTAC
  • Peptide(s) is cross-validated by a different peptide or orthogonal technique
  • Suggested spike-in level for single-point calibration in specified matrix

Single-Point Calibration

The traditional method for absolute quantification employing mass spectrometry is reliant upon the production of an external protein standard curve and spike-in of the equal amount of a stable isotope-labeled standard to the samples.

In this method, the stable isotope-labeled standard is utilized exclusively for the purpose of normalization and does not need to be precisely quantified.

The use of an external standard curve needs multiple further LC-MS investigations to be performed and the precision of this type of assay is highly reliant upon the quality of the protein utilized for the standard curve.

The concentration of the standard must be accurately calculated employing a highly quantitative assay. The standard protein also must be well characterized in regards to the modifications and structure, which should be the same as for the endogenous protein of interest.

A more popular and high-throughput method for absolute quantification is the introduction of stable isotope-labeled standards with precisely established concentrations. The quantification of the endogenous protein is then achieved by the direct comparison of the ratio between labeled and unlabeled peptides.

Dependent on the range of the linear response, the slope and the intercept, this technique can enable the use of single-point calibration.

QPrEST+ standards are ideally suited for use in single-point calibration studies because they are quantified beforehand with great accuracy and comprise of one or multiple validated peptide(s). Furthermore, the QPrEST+ standards are also supplied with suggested spike-in levels that provide labeled:unlabeled ratios close to one in a standard sample, which is preferred to attain strong repeatability and accuracy for single point calibrations.

As QPrEST+ peptides have a validated linear response, they also tend to offer beneficial quantitative results when analyzing quite large off-ratios (as long as both the light and heavy signals are not too near to the lower quantification limit).

QPrEST Validation Example

A QPrEST (QPrEST22551) for Leucine-rich alpha-2-glycoprotein-1 (LRG1/A2GL) (Figure 3) was serially diluted in three steps and spiked into pooled human plasma at eleven varying concentrations.

The samples were reduced, alkylated, and digested with trypsin and were then evaluated by LC-MS/MS in parallel reaction monitoring (PRM) mode utilizing a Thermo Q Exactive Plus MS Instrument.

Two tryptic peptides (VAAGAFQGLR and DLLLPQPDLR) with three transitions each were assessed with an external tool from CPTAC to produce plots.

Linear response curves were created from 2 to 13,300 fmol/µL plasma with equivalent slopes near to one and intercepts near to zero for both of the peptides (Figure 4). The peptides demonstrated similar linear responses which validated each other.

The LLOQ was established to be 2 fmol/µL. The plasma level of LRG1/A2GL was calculated as 300 fmol/µL in a healthy plasma pool. Repeatability for LRG1/A2GL peptides, DLLLPQPDLR and VAAGAFQGLR, was studied in samples with low, medium, and high spike-in levels. They were prepared and evaluated five times on five individual days (Figure 5).

The three alternate spike-in levels offered results that were similar and the repeatability was well within the CPTAC criteria of variation (less than 20%).

Amino acids sequence for LRG1/A2GL: The Leucine-rich alpha-2-glycoprotein-1 (LRG1/A2GL) consists of 347 amino acids and the sequence is outlined above. The QPrEST22551 (green font) cover amino acids 193-326. The QPrEST22551 tryptic peptides DLLLPQPDLR and VAAGAFQGLR (underlined) were included in the CPTAC validation.

Figure 3. Amino acids sequence for LRG1/A2GL: The Leucine-rich alpha-2-glycoprotein-1 (LRG1/A2GL) consists of 347 amino acids and the sequence is outlined above. The QPrEST22551 (green font) cover amino acids 193-326. The QPrEST22551 tryptic peptides DLLLPQPDLR and VAAGAFQGLR (underlined) were included in the CPTAC validation. Image Credit: Atlas Antibodies

Figure 4. Response curves for LRG1/A2GL: Linear response curves for Leucine-rich alpha-2-glycoprotein-1 (LRG1/A2GL) peptides DLLLPQPDLR (upper images) and VAAGAFQGLR (bottom images) derived after protease digestion of QPrEST22551. Image Credit: Atlas Antibodies

Repeatability study for LRG1/A2GL: Repeatability study for LRG1/A2GL peptides DLLLPQPDLR (left) and VAAGAFQGLR (right). Samples with high (13 pmol/µL plasma), medium (1.5 pmol/µL plasma) and low (0.02 pmol/µL plasma) spike-in levels were prepared five separate days and analyzed in triplicate injections.

Figure 5. Repeatability study for LRG1/A2GL: Repeatability study for LRG1/A2GL peptides DLLLPQPDLR (left) and VAAGAFQGLR (right). Samples with high (13 pmol/µL plasma), medium (1.5 pmol/µL plasma) and low (0.02 pmol/µL plasma) spike-in levels were prepared five separate days and analyzed in triplicate injections. Image Credit: Atlas Antibodies

Summary

  • QPrESTs are stable isotope-labeled protein standards for absolute quantification utilizing mass spectrometry
  • The QPrEST+ protein standards are widely available and include peptides that have been verified in line with the CPTAC assay criteria
  • The QPrEST+ protein standard offers quantotypic peptides similar to stable isotope-labeled full-length proteins
  • QPrEST+ displays good linear responses for quantotypic peptides and are suitable for single-point calibration

References and Further Reading

  • Bantscheff M, Lemeer S, Savitski MM, Kuster B. (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404, 939-965
  • Zeiler M, Straube WL, Lundberg E, Uhlén M, Mann M. (2012) A Protein Epitope Signature Tag (PrEST) library allows SILIAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines. Mol Cell Proteomics 11, O11 009613
  • MacCoss MJ. Mass Spectrometry Signal Calibration for Protein Quantitation. Technical note from Cambridge Isotope Laboratories.

Atlas Antibodies

The QPrEST+ products will be available early 2020, please browse the current catalog of 20.000 QPrESTs at 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: Jan 30, 2020 at 2:39 PM

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