Qcal peptide mix

External spike-in normalization is targeted at correcting for unwanted technical variation introduced throughout the experiment. Therefore, an external reference standard is spiked into each sample at a known concentration. Here, QCAL peptide mix (Sigma Aldrich), designed as universal MS standard, was employed as reference. The normalization was performed in Proteome Discoverer. Reference protein abundance is calculated for each sample determining the maximum abundance in all samples. The normalization factor results as the factor of the maximum reference protein abundance of all samples and the individual abundance of the corresponding sample: $${\tilde{X}}_{\mathit{ij}}=\frac{X_{\mathit{ij}}\ast \underset{1\le i\ge M}{max}\left(X_{i,QCAL}\right)}{X_{i,QCAL}},$$ where X_i,QCAL denotes the QCAL protein abundance in sample i.