Proof of the quantitative potential of immunofluorescence by mass spectrometry
Maria I Toki1, Fabiola Cecchi2, Todd Hembrough2
1Department of Pathology, Yale University Medical School, New Haven, CT, USA.
Insights
Quantitative immunofluorescence (QIF) can achieve mass spectrometry (MS)-level protein quantification. Optimizing antibody concentration via signal-to-noise ratio standardization allows QIF to accurately measure absolute protein levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Immunohistochemistry (IHC) is a standard method for protein expression analysis in patient tissues.
- IHC is limited by variability, affecting sensitivity, specificity, and reproducibility, yielding semi-quantitative results.
- Mass spectrometry (MS) is the gold standard for protein quantification due to its high sensitivity, specificity, and objective molecular measurement.
Purpose of the Study:
- To demonstrate that standardized quantitative immunofluorescence (QIF) can achieve quantitative protein measurements comparable to MS.
- To establish a method for standardizing QIF to MS for absolute protein concentration assessment.
Main Methods:
- Quantitative immunofluorescence (QIF) was used to measure epidermal growth factor receptor (EGFR) expression in 15 cell lines.
- EGFR was quantified across a range of primary antibody concentrations, including the optimal signal-to-noise ratio concentration determined by titration.
- QIF measurements were compared against absolute EGFR concentrations determined by Liquid Tissue-selected reaction monitoring mass spectrometry (MS).
Main Results:
- The optimal signal-to-noise ratio concentration for the primary antibody yielded the best agreement between QIF and MS.
- A strong linear regression (R²=0.88) was observed between QIF and MS measurements when using the optimized antibody concentration.
- Standardization of QIF via signal-to-noise ratio calculation enabled reproducible assessment of absolute protein concentration.
Conclusions:
- Quantitative immunofluorescence (QIF), when optimized and standardized, can provide quantitative protein measurements comparable to mass spectrometry (MS).
- Calculating the signal-to-noise ratio for primary antibody titration is crucial for standardizing QIF to MS.
- This standardized QIF approach allows for linear and reproducible determination of absolute protein concentrations.
Abstract:
Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R2=0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.
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