Metalloproteome factory: from crude extract to identification of metalloproteins via 3-dimensional HPLC-ICP-sfMS

Metalloproteins are essential for a vast array of biological processes in all living organisms. Identification of multiple metalloproteins from native expression (“metalloproteomics”) based on a single chromatography has low resolution and/or sensitivity, while classical protein purification lacks the versatility required for purifying multiple proteins for proteomics. We present a new metalloproteomics method based on three dimensions of liquid chromatography hyphenated with inductively coupled sector-field mass spectrometry (ICP-sfMS): anion exchange chromatography (AEC) followed by either chromatofocusing (CF) or hydrophobic interaction chromatography (HIC), and finally size exclusion chromatography (SEC). We used this 3D technique to purify naturally abundant metalloproteins from crude (mainly) plant, bacteria and animal extracts to a quantity and purity sufficient for protein identification and quantification by mass spectrometry (Tims-TOF-MS). Sensitivity and signal/background ratio were sufficient to identify proteins at concentrations < 1 nM and total amounts < 1 pM in the final SEC.