MRM Assays Advantages

Our MRM assays (existing and customized) have several significant advantages over other protein/biomarker quantification methods, including HPLC and antibody based ELISA assays.

 

• Short assay development time – 6 to 10 weeks.
• No antibody development required
• Highly Multiplexed (10s to 100s)
• Absolute quantification
• Absolute molecular specificity
• Rapid (30 min. runs)
• Sensitive (low ng to pg protein/ml) – in undepleted plasma
• Robust & reproducible
• Minimal sample volumes (5-20ul)

 

While we recognize that other companies appear to develop similar MRM-based quantitative proteomic assays a few key differences help distinguish MRM Proteomics (i.e., interference testing, no depletion or enrichment, and rigorous quality control).  These are discussed below:

 

Protein Quantitation without Depletion or Enrichment
• improves reproducibility, diminishes costs, prevents target analyte loss
• High sensitivity (ng – pg protein/ml) in undepleted plasma

 

SIS internal standard peptides
• to optimize performance parameter achieving highest sensitivity
• to obtain superior specificity, reproducibility and accuracy
• to compensate for losses

 

Rigorous Chemical Interference Testing
• to approach absolute specificity
• to obtain reliable quantification

 

The use of concentration-balanced SIS peptides
• to reduce the analytical variation of the assay
• to maximize the linear range of the MRM assay
• to improve the quantitative accuracy

 

 

No Depletion or Enrichment

The lower limits of quantitation in our MRM methods are not achieved with preanaytical immunoaffinity depletion or antibody-based enrichment, as is typically employed in bottom-up proteomic workflows.

 

Doing depletion or enrichment adds additional cost to assays, can diminish sample throughput, can increase the %CV by
a factor of 7% to 25% (in part because depletion cartridges and columns are usually used multiple times), and also can remove clinically-relevant high-abundance proteins.  We and others have also observed that depletion can also remove low molecular weight and low abundance proteins of interest.

 

 

Stable Isotope-labeled Standard (SIS) Peptides

The use of SIS peptides not only allows "absolute" quantitation (i.e, the determination of protein concentrations instead
of relative "fold-changes" in relative quantitative techniques), but also provides an internal standard in every spectrum.  This plays an indispensable role in interference testing (discussed below), while correcting for any sample losses incurred during analysis following its addition.

 

The MRM-MS with SIS peptide approach has been billed the “gold standard” for MS-based protein quantitation (Ong S et al. Nature Chemical Biology. 1 `05 252).  The directed quantitative approach is capable of sensitive (attomole level) and "absolute" determination of peptide, and proteins by inference, concentrations across a concentration range exceeding 104, as demonstrated in recent publications.

 

 

Chemical Interference Testing

We perform rigorous interference screening on all samples prior to protein quantitation.  The testing is conducted on the same biological matrix as the sample and is necessary due to the inherent complexity of the biofluid.

 

The thousands of proteins present in unfractionated plasma or other biofluid samples can cause overlapping isotopic distributions in the mass transmission windows, which can invalidate the accuracy of the quantitative technique.

 

We perform such evaluations using the signals from the synthetic (SIS) and the endogenous (natural, NAT) peptides The SIS and NAT peptide signals are rigorously inspected and only those that:

• co-chromatograph
• exhibit similar peak shapes
• lack non-specific, co-eluting ions pass the first test

 

A second test involves average relative ratio calculations of the MRM transitions between SIS in buffer, SIS in biofluid, and NAT in biofluid.  These must exhibit <20% variability to be qualified as being interference-free.

 

While some MRM assay service providers claim to perform interference testing, such determinations are based solely on retention time alignment, which could cause peptides with co-eluting ions to be selected as targets and lead to inaccurate quantitative measurements.  Accurate interference testing cannot be done without the use of SIS peptides.

 

 

Concentration-balanced SIS peptides

An additional difference between our approach and the other MRM-based methods, even those using SIS peptides, is that ours are developed using a concentration-balanced, rather than an equimolar, mixture of SIS peptides.

 

This ensures that the SIS peptide internal standards are as close as possible to those of the endogenous protein concentration in the biological sample during LC-MRM/MS analyses.

 

Balancing the ratios allows us to:

• minimize the analytical variation between analyses
• improve the quantitative accuracy
• maximize the linear range of the assays

 

In so doing, CVs of <10% are routinely obtained in our intra-/inter-day analyses and within a given concentration level in the per-peptide calibration curves.

MRM Proteomics – Protein Quantification and Biomarker Discovery by Multiple Reaction Monitoring