Remarkable effect of mobile phase buffer on the SEC-ICP-AES derived Cu, Fe and Zn-metalloproteome pattern of rabbit blood plasma.
The development of an analytical method to quantify the major Cu, Fe and Zn-containing metalloproteins in mammalian plasma has been recently reported. This method is based on the separation of plasma proteins by size exclusion chromatography (SEC) followed by the on-line detection of the metalloproteins by an inductively coupled plasma atomic emission spectrometer (ICP-AES). To assess whether the mobile phase buffer can affect the SEC-ICP-AES-derived metalloproteome pattern, thawed rabbit plasma was analyzed using phosphate buffered saline (PBS)-buffer (0.15 M, pH 7.4), Tris-buffer (0.1 and 0.05 M, pH 7.4), Hepes-buffer (0.1 M, pH 7.4) or Mops-buffer (0.1 M, pH 7.4). In contrast to the Cu-specific chromatograms, the Fe and Zn-specific chromatograms that were obtained with Tris, Hepes and Mops-buffer were considerably different from those attained with PBS-buffer. The Tris, Hepes and Mops-buffer mediated redistribution of ~25% plasma Zn(2+) from <100 kDa to >100-600 kDa plasma proteins and to a smaller extent to a <10 kDa (Tris)(2)Zn(2+)-complex can be rationalized in terms of the abstraction of Zn(2+) from the weak binding site on albumin. In contrast, only Hepes and Mops-buffer redistributed ~20% of plasma Fe(3+) from the <100 kDa to the >600 kDa elution range. Based on these results and considering that the utilization of PBS-buffer has previously resulted in the detection of a number of Cu, Fe and Zn-containing metalloentities in rabbit plasma that was most consistent with literature data, this mobile phase buffer is recommended for metallomic studies regarding mammalian blood plasma.
Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada.
Elham Zeini
EZ
Jahromi
Wade
W
White
Qiao
Q
Wu
Raghav
R
Yamdagni
Jürgen
J
Gailer
D000818:Animals; D002021:Buffers; D002850:Chromatography, Gel; D003300:Copper; D006531:HEPES / Q000737:chemistry; D007501:Iron; D008667:Metalloproteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D019216:Metals, Heavy / Q000097:blood / Q000737:chemistry; D009025:Morpholines / Q000737:chemistry; D010949:Plasma / Q000378:metabolism*; D020543:Proteome; D011817:Rabbits; D013054:Spectrophotometry, Atomic; D014325:Tromethamine / Q000737:chemistry; D015032:Zinc
D002021:Buffers; D008667:Metalloproteins; D019216:Metals, Heavy; D009025:Morpholines; D020543:Proteome; D014325:Tromethamine; C008550:morpholinopropane sulfonic acid; D003300:Copper; D007501:Iron; D015032:Zinc; D006531:HEPES