Affinity purification contaminants identified by cryo-EM and mass spectrometry

Affinity chromatography is a powerful and therefore popular method for the purification of proteins for structural studies. The success of the technique relies on the specificity of the interaction between the target protein and the affinity resin. Here, we present the identification of two protein contaminants isolated from HEK293 cell lysate following affinity purification of twin Strep-tagged or FLAG-tagged proteins. The contaminants were identified as human propionyl-coenzyme A carboxylase and protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) via a combination of cryogenic electron microscopy (cryo-EM) data processing and proteomic analyses. This report serves to illustrate how these contaminants may appear in cryo-EM datasets and to highlight the paramount importance of affinity chromatography resin specificity for efficient protein purification.