期刊
SCIENTIFIC REPORTS
卷 7, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/srep45570
关键词
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资金
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/M018725/1, BB/L009501/1]
- BBSRC CASE studentship [BB/J012874/1]
- Biotechnology and Biological Sciences Research Council [1366244, BB/M018725/1] Funding Source: researchfish
- Medical Research Council [MR/M009114/1] Funding Source: researchfish
- BBSRC [BB/M018725/1] Funding Source: UKRI
- MRC [MR/M009114/1] Funding Source: UKRI
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-kappa 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.
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