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Online Miniaturized Asymmetrical Flow Field-Flow Fractionation and Inductively Coupled Plasma Mass Spectrometry for Metalloprotein Analysis of Plasma from Patients with Lung Cancer
journal contribution
posted on 2016-09-17, 00:00 authored by Jin Yong Kim, Heung Bin Lim, Myeong Hee MoonMetalloproteins (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.