Sensitive determination of metalloprotein in salt-rich matrices by size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry.

Metalloproteins play crucial and distinct roles in a variety of biological processes that rely heavily on the metal ions and various proteins. However, there is still a lack of method for rapid analysis of metalloproteins in complex samples, especially in salt-rich matrices. In this study, a sensitive method for separation and determination of metalloproteins in salt-rich matrices was developed based on the size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry (SEC-ICP-MS), combining with the high matrix introduction (HMI) mode, which is quite essential for biological system. The separation conditions of the SEC-ICP-MS system were optimized by using four iodine labeled proteins with different molecular weights, including bovine serum albumin (BSA, 66.0 kDa), ovalbumin (OVA, 44.0 kDa), carbonic anhydrase (CA, 29.0 kDa) and ribonuclease A (RA, 13.7 kDa). After optimization, four iodine labeled proteins and iodine ions were successfully separated within 30 min by using 10 mmol/L HEPES and 40 mmol/L NaSO (pH=7.0) as mobile phase and a linear relationship between log molecular weight and retention time was established. The relative standard deviations (RSDs, n = 5) of the retention time and peak areas for the four iodine labeled proteins were in the range of 0.2-0.9% and 3.3-7.7%, respectively, suggesting good precision and repeatability. Then the proposed method was successfully applied to the rapid separation and detection of lead-binding proteins in real biological tissue samples.