DOSCATs: Double standards for protein quantification

Richard J Bennett,Philip Brownridge,Claire E Eyers,Robert J Beynon,Deborah M Simpson,Stephen W Holman,Sheila Ryan,John Colyer

doi:10.1038/srep45570

Richard J Bennett, Philip Brownridge + Show 6 more

Open Access

https://doi.org/10.1038/srep45570

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Abstract

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-κB 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.

Highlights

Many papers that report sqWB data do not include exhaustive data that defines the specificity of the antibody-antigen interaction, linearity of response or evidence that the immunoreactive band is the target antigen
In pursuit of reliable approaches to quantitative western blotting, there is merit in comparison of such approaches with an orthogonal method, such as those based on mass spectrometry
For DOSCATs, epitopes for a small panel of antibodies are inserted into the protein in addition to Q-peptides so that the standard can be deployed across both mass spectrometry (MS) and quantitative western blotting (QWB) workflows

Summary

Introduction

Many papers that report sqWB data do not include exhaustive data that defines the specificity of the antibody-antigen interaction, linearity of response or evidence that the immunoreactive band is the target antigen It is common practice in publication of sqWB results to crop western blot images to the region of interest, obscuring other regions of cross-reactivity. It would be ideal if calibration standards were capable of deployment across both MS and QWB workflows This crossover would allow for validation of the orthogonal techniques and comparison of data between the two most common quantitative techniques. DOSCATs concatenate epitope sequences from one or more proteins recognised by multiple antibodies They embed quantotypic peptides (Q-peptides) for MS quantification of the same proteins (Fig. 1) and act as a single multiplexed standard that can be used for MS-based or QWB quantification. The aim of this work is to demonstrate that DOSCATs can be used as a calibration standard across both SRM and QWB workflows to deliver equivalent quantitative results

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