Double standards in quantitative proteomics: Development of calibrators for multiplexed quantitative western blotting or mass spectrometry

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology


Biological research is a largely quantitative science, and in biomolecular sciences, there is an ever-present need to be able to measure the amounts of specific molecules, and changes in their abundance, in a highly sensitive fashion. Moreover, the biomolecules have to be measured in a background of many tens of thousands of similar biomolecules, and thus, the measurements must be selective as well as sensitive.

There are two analytical approaches that are commonly used to count the numbers of biomolecules in a system. One relies on a very expensive and extremely sophisticated instrument, a mass spectrometer, and requires highly skilled specialist staff to deliver the very complex assays that are developed for this platform. The second approach uses very inexpensive laboratory equipment (likely to be found in almost every lab) and relies on antibodies to 'find' and measure the biomolecule of interest - a magic bullet for measurement. However, antibody methods, as practiced in many labs, are not really quantitative. They rely on separation of biomolecules in a sloppy gel, and then 'blotting' the biomolecules onto a teflon membrane before using antibodies to 'find; the biomolecule of interest -so called 'western blots'. Western blots are messy, highly variable and error prone - as has been commented, westerns can be good, bad, or ugly!

To circumvent the problems of manual western blots, an affordable commercially available instrument has come to market that, in our hands, is capable of performance at least as good as a mass spectrometer. We believe that instrument such as this has huge potential to bring immunological quantification via westerns to the same standard as mass spectrometry.

In our experience, the two methods (automated antibody detection or mass spectrometry) can be made to perform equally well, but in some circumstances, the results do not agree. One of the problems is that we lack suitable standards for both methods, which prevents direct comparison. To solve this problem, we have invented the concept of artificial proteins (DOSCATs) , never previously seen on the planet, that are created through gene design to perform a specific function - providing standard proteins that can be used equally well for immunological or mass spectrometric quantification. This innovative concept will create a seamless connection between the two routes to quantification, and at the same time, will enhance the quantification to permit accurate counting of the numbers of specific molecules in a biological sample.

Because this is a technological development application, the test systems that we will use are selected because we know them well, and know how they behave in natural systems. This reduces uncertainty when we come to perform quantification by mass spectrometry or western blotting. We will develop DOSCATs that address diverse areas such as mastitis in dairy cattle (which has both welfare and commercial consequences), separate quantification of very similar biomolecules (called isoforms) in a project that aims to understand and develop strategies for enhanced rodent control and a programme that has the goal of providing precise quantitative data to computational biologists who are trying to build models of living process.

Technical Summary

We have previously demonstrated that it is possible to sue de novo gene design to create genes that when transcribed and translated generate novel proteins that can then be used in quantitative mass spectrometry. A key example are QconCATs, concatenations of tryptic peptides that are stable isotope labelled and which when proteolysed, generate a stoichiometrically equal mix of over 50 standard peptides for selected reaction monitoring mass spectrometric analyses for absolute quantification of protein abundance.

In the proposal, we will apply equivalent thinking and approaches to the development of novel proteins that can be used to enhance the quality of quantitative western blotting and quantitative mass spectrometry in a multiplexed and parallel format. These new proteins, called DOSCATs, will contain epitopes that are derived from multiple proteins and at the same time, are interspersed by peptides that act as mass spectrometric standards. These 'double standards' can thus be used to support either quantitative platform, or ensure clarity of the relationship between the two methodologies.

In this proposal, we will develop the concept and application of DOSCATs, using a limited number of well understood model systems, to optimise gene design, expression and deployment. We will make particular use of the DOSCATs in automated western blotting, a technique that promise to supplant all manual based quantification, as it delivers superior performance in terms of sensitivity and intrinsic variance. We will also used intelligent gene design to explore factors that might influence epitope accessibility or excision efficiency.

At the end of the programme, we aim to gain sufficient technical information to understand how to deploy DOSCATs in biological research, and to generate adequate preliminary data to explore the IP potential of the technology.

Planned Impact

This project will validate new quantitative technologies for protein research (cell biology, systems biology, proteomics) which builds on the previous patented IP for QconCAT technology. By developing new, multiplexed, dual purpose standards, we can ensure greater comparability between research groups, and ensure that comparability between mass spectrometric and immunological methods are maintained.

The beneficiaries of these technologies and products are:

(1) the life science and medical research community in academia (globally) and industry (pharmaceutical, biopharmaceutical, agrichemical, diagnostic, analytical/contract research sector).

The project will benefit these groups by providing the means of performing quantitative protein measurements with analytical techniques familiar to the research community (western blot). End users will benefit from an increase in the quality of data generated by research (the capture of analytical measurements in absolute chemical units), an increase in productivity of research (internal standards will reduce variance in data collected longitudinally, and permit detection of statistically relevant changes in smaller sample groups), and will increase the productivity of whole field of research through the exchange of information in a common (chemical) currency.

(2) Other beneficiaries include companies in the supply and support of this new market (see letter of support from project partner, Badrilla). These organisations will experience growth in revenue and staff numbers arising from the manufacture of new products for a new market. More indirectly, the project will benefit marketing, conference and social media companies as the full exploitation of this capability is realised.

(3) Additional commercial and patient/public beneficiaries are likely as the technology is applied to translational topics. These areas are likely to include contract research services, diagnostic assay discovery & validation, and perhaps biomarker measurement in clinical trials. Patients and the public will benefit indirectly from the diagnostics and therapeutics that are developed using these technologies. In a small number of cases, quantitative western blotting may represent the diagnostic test itself (Lyme's disease, HIV, CJD) and thus beneficiaries will include the service providers (NHS, commercial) and patients.

(4) Finally, the UK economy will be a beneficiary as these (largely) UK companies increase activity to create and serve the new market. The value of other markets in diagnostics and services has not been calculated at this point, but is likely to be higher than that of fundamental research.


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Description We have proven that it is possible to design, produce and use DOSCATs that can act as double, multiplexed standards in proteomics. This work has now been published in a leading journal. The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-?B pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.

We have also used the DOSCAT approach in a separate study, quantifying proteins in CSF. Streptococcus pneumoniae is responsible for diseases causing major global public health problems, including meningitis, pneumonia and septicaemia. Despite recent advances in antimicrobial therapy, pneumococcal meningitis remains a life-threatening disease. Furthermore, long-term sequelae are a major concern for survivors. Hence, a better understanding of the processes occurring in the central nervous system is crucial to the development of more effective management strategies. We used mass spectrometry based quantitative proteomics to identify protein changes in cerebrospinal fluid from children with Streptococcus pneumoniae infection, compared with children admitted to hospital with bacterial meningitis symptoms but negative diagnosis. Samples were analysed, by label free proteomics, in two independent cohorts (cohort 1: cases (n = 8) and hospital controls (n = 4); cohort 2: cases (n = 8), hospital controls (n = 8)). Over 200 human proteins were differentially expressed in each cohort, of which 65% were common to both. Proteins involved in the immune response and exosome signalling were significantly enriched in the infected samples. For a subset of proteins derived from the proteome analysis, we corroborated the proteomics data in a third cohort (hospital controls (n = 15), healthy controls (n = 5), cases (n = 20)) by automated quantitative western blotting, with excellent agreement with our proteomics findings.
Exploitation Route We have evaluated DOSCATs for IP and have been informed by independent patent agents that the IP would be difficult to protect. We have therefore adopted a strategy of working with BAdrilla to access major antibody companies who would be interested in multiplexed calibrators. Further funding has been attained through a KTP grant between Liverpool and Badrilla.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

Description DOSCATS are now being developed for commercial use, using a KTP fellowship with BAdrilla. This has focused on the use of cell-0free synthesis and optimsiation of blotting and multiplexed protocols. This work is ongoing.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description KTP Associate
Amount £180,000 (GBP)
Funding ID KTP 010806 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 02/2017 
End 01/2019
Description STEM 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Built a strong relationship with Winstanley College, leading to joint grants from RSC and RSoc.
Year(s) Of Engagement Activity 2014,2015,2016