Trypsin digestion to prevent acid induced precipitation of metalloproteins for accurate ICP-MS analysis: Nitrogenase...

Accurate determination of metallation states in metalloenzymes is essential for correlating metal content with enzymatic function. Inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (ICP-OES) are among the most sensitive and practical techniques for this purpose. However, complete digestion of protein samples is critical for accurate results, and the use of acid often leads to protein precipitation before the metals are fully released into solution. Here, we present a case study using the nitrogenase protein NifDK, which contains both Fe and Mo, to demonstrate that trypsin digestion can prevent acid-induced precipitation. A 1:1 (w/w) ratio of protein to trypsin in Tris buffer at 37 °C overnight results in complete digestion of NifDK. Upon acid addition, a homogeneous solution is obtained without precipitation. This approach yields highly reproducible ICP-MS data. Further improvements include performing protein quantification on the exact vial used for ICP-MS analysis, applying a drift correction to the ICP-MS data, and using nitric acid with 0.1 % (v/v) HF for accurate molybdenum quantification.