Quantitative analysis of proteins via sulfur determination by HPLC coupled to isotope dilution ICPMS with a hexapole collision cell.
Quantitative analysis of proteins is an essential part and also constitutes a major challenge in modern proteomics. Quantification of proteins by inductively coupled plasma mass spectrometry (ICPMS) offers an alternative method for quantitative proteomics. In this study, we developed a method of absolute quantification of proteins via sulfur by size exclusion chromatography (SEC) coupled to ICPMS with a collision cell (ICP-CC-MS) and postcolumn isotope dilution. Bovine serum albumin (BSA), superoxide dismutase (SOD), and metallothionein-II (MT-II) served as model proteins. Enriched 34S, 65Cu, and 67Zn isotopic solutions were continuously mixed with the eluate from the SEC. Oxygen was added as a reactive gas into the collision cell where sulfur reacts with oxygen to form sulfur-oxygen ion, the ratio of 32S16O(+)/34S16O(+) thus representing 32S(+)/34S(+). The absolute quantity of proteins could be calculated by the isotopic dilution equation and the content of sulfur in the proteins. The detection limits for BSA, SOD, and MT-II are 8, 31, and 15 pmol, respectively. The relative standard deviations for the proteins are less than 3%. The ratios of S/Cu and S/Zn in the proteins were also determined. The quantitative method was validated by comparing with gravimetric results.
Wang
Meng
M
Key Laboratory of Nuclear Analytical Techniques and Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China.
Feng
Weiyue
W
Lu
Wenwei
W
Li
Bai
B
Wang
Bing
B
Zhu
Motao
M
Wang
Yun
Y
Yuan
Hui
H
Zhao
Yuliang
Y
Chai
Zhifang
Z
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D013455:Sulfur / Q000032:analysis*
D007554:Isotopes; D011506:Proteins; D013455:Sulfur