Three representative protein kinases with different substrate preferences, ERK1 (Pro‐directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T‐cell leukemia). Here, WEE1 is linked to breast carcinoma.