DOSCATs: Double standards for protein quantification

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.

Median based absolute quantification of proteins using Fully Unlabelled Generic Internal Standard (FUGIS)

“…MBAQ workflow for absolute quantification MBAQ (for mean based absolute quantification) workflow relies on a recombinant protein standard consisting of concatenated peptides, whose sequences emulate physicochemical properties shared by typical proteotypic peptides. Its tryptic cleavage produces peptides in exactly equimolar concentration 16,21,35 , as evidenced by the time course and relative abundances of rendered peptides 22 . Therefore, peptides concentration could be inferred from the known molar abundance of the chimera.…”

“…AQUA [13] , an absolute quantification method uses a set of isotopically labelled synthetic peptide standards mimicking the proteotypic peptides from endogenous proteins. With advances in gene synthesis, techniques like QconCAT [14] , PSAQ [15] , PrEST [16] , PCS [17] , MEERCAT [18] , DOSCAT [19] , and GeLC-based MS Western [20] were developed which uses isotopically labelled protein chimeras that, upon proteolytic cleavage, produce the desired proteotypic standards. Unlike the aforementioned strategies, MS Western uses multiple proteotypic peptides per protein, which provides concordant quantification by controlling the ratio of abundances of peptide peaks.…”

“…AQUA 15 uses a set of isotopically labelled synthetic peptide standards identical to proteotypic peptides from endogenous proteins. Alternatively, QconCAT 16 , PSAQ 17 , PrEST 18 , PCS 19 , MEERCAT 20 , DOSCAT 21 and GeLC-based MS Western 22 employ metabolically labelled protein chimeras that, upon proteolytic cleavage, produce the desired peptide standards. MS Western relies on quantifying multiple proteotypic peptides per protein and validates the concordance of proteins determinations by monitoring the intensity ratios between XIC peaks of standards and corresponding endogenous peptides.…”

MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification

“…The ease of delivery of adequate quantities of labeled protein allows for cycles of exploration and refinement. Other concatamers have been developed for standardization of chromatography elution ( 36 ) and as hybrid standards for quantification of proteins by mass spectrometry and quantitative Western blotting ( 37 ). As more complex designs are conceived, it is increasingly attractive to be able to express such proteins free from issues of cellular toxicity/failure to express and proteolytic degradation.…”

FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

“…Alternatively, an additional (secondary) peptide concatenated standard (PCS) was used to quantitatively characterize multiple primary PCSs. 18 Also, a QconCAT chimera could include the sequence of [Glu1]-Fibrinopeptide B 19 that enabled its one-peptide quantification, although synthetic peptide standards should be used with caution. 20…”