Simultaneous determination of nitrated and oligomerized proteins by size exclusion high-performance liquid chromatography coupled to photodiode array detection.
Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking.
Multiphase Chemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 Mainz, Germany.
Fobang
F
Liu
Multiphase Chemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 Mainz, Germany.
Kathrin
K
Reinmuth-Selzle
School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China; Multiphase Chemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 Mainz, Germany.
Senchao
S
Lai
Division 1.5 Protein Analysis, Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
Michael G
MG
Weller
Multiphase Chemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 Mainz, Germany.
Ulrich
U
Pöschl
Multiphase Chemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 Mainz, Germany; Institute for Organic Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, 55128 Mainz, Germany; Institute for Inorganic and Analytical Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, 55128 Mainz, Germany. Electronic address: kampfc@uni-mainz.de.
Christopher J
CJ
Kampf
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D009566:Nitrates / Q000737:chemistry*; D009585:Nitrogen Dioxide / Q000737:chemistry; D010126:Ozone / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013774:Tetranitromethane / Q000737:chemistry
D009566:Nitrates; D011506:Proteins; D010126:Ozone; D013774:Tetranitromethane; D009585:Nitrogen Dioxide
HPLC-DAD; Nitrotyrosine; Protein nitration degree; Protein oligomer analysis; Size exclusion chromatography