Online Miniaturized Asymmetrical Flow Field-Flow Fractionation and Inductively Coupled Plasma Mass Spectrometry for Metalloprotein Analysis of Plasma from Patients with Lung Cancer
2016-09-17T00:00:00Z (GMT) by
Metalloproteins (metal-binding proteins) refer to proteins containing metal ion cofactors. The importance of these proteins has increased owing to their involvement in many biological processes. Here, we introduce an analytical platform based on online coupling of miniaturized asymmetrical flow field-flow fractionation (mAF4) and inductively coupled plasma mass spectrometry (ICPMS) for size separation of proteins followed by the detection of metals associated with plasma metalloproteins. Not only did the mild separation of mAF4 get carried out in a biological buffer solution to minimize disruption of the metal-complex structure but free metal ions and salts from complicated biological samples were also removed during separation by crossflow. The relative quantities of metalloproteins detected by mAF4-ICPMS between plasma samples from patients with lung cancer and healthy controls were compared by determining the peak areas of detected elements and retention times; among these, 7 (55Mn, 60Ni, 63Cu, 66Zn, 90Zr, 127I, and 137Ba) out of 16 elements showed substantial changes in patients with lung cancer. For the quantitative comparison of metalloproteins, protein fractions during mAF4 were collected and analyzed by nanoflow liquid chromatography–tandem mass spectrometry using isotope-coded carbamidomethylation. Quantitative analysis showed that some metalloproteins associated with 55Mn, 60Ni, 63Cu, and 66Zn exhibited changes similar to those in patients. These findings demonstrated the potential of mAF4-ICPMS as a powerful high-speed screening method for targeted metalloproteins related to diseases.