Advances in the quantification of protein microarrays

•Lysate microarrays were used for the analysis of signaling events. The authors carefully evaluated the used antibodies by different means.•The paper describes the validation of binders for different applications and thereby, providing a workflow that other researchers can follow.•The paper focuses on the standardized evaluation of peptide microarrays.•The paper describes a way to obtain quantitative mass spectrometric data and can be applied as guideline for protein/peptide microarrays.

The determination of the protein composition is a challenging task, yet a valuable endeavor. Information that lies within the composition can help predicting pathogenic mechanisms and diseases. Therefore, a mandatory requirement is the accurate quantification. Protein microarrays are a promising alternative to complement mass spectrometry (MS) techniques. Nevertheless, effects in microarray production and assay development may lead to altered signal intensities. Techniques like NormaCurve provide a more robust quantification by taking the immobilized protein content into account. Printing dilution series can be used to construct a non-linear standard curve to estimate the amount of bound IgG, which helps to overrule the detection range of one order of magnitude. Standard protocols based on projects like the ‘minimum information about a proteomics experiment’ will help to improve quality and overall comparability.