Naslov
Determination of dithiothreitol in complex protein mixtures by HPLC-MS

Autori
Cindrić, Mario ; Čepo, Tina ; Marinc, Sabina ; Paškvan, Ivan ; Mijić, Ivana ; Bindila, Laura ; Peter-Katalinic, Jasna

Izvornik
Journal of separation science (1615-9306) 31 (2008), 20; 3489-3496

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
HPLC-APCI-MS/MS; oxidized dithiothreitol; protein refolding; recombinant proteins

Sažetak
Dithiothreitol (DTT) and other reducing agents are typically used in refolding processes of recombinant human proteins during their purification from inclusion bodies. Due to its toxicity, it is essential to monitor the clearance of DTT throughout the analytical flow from the refolding phase to the final formulated product. Here we report a direct, simple, and fast liquid chromatography method using UV and tandem mass spectrometry (MS/MS) detection for DTT evaluation in complex protein mixtures. In aqueous solution DTT exists as an equilibrium mixture of the oxidized and the reduced form (H2DTT double left right arrow DTTox) and the quantitation tools should therefore be applicable to both forms in a single step or in multiple steps. Oxidation of DTT with aqueous copper(II) nitrate trihydrate solution was introduced to determine a single oxidized compound, i.e. DTTox. Proteins and other components of high molecular masses were separated from DTTox by ultrafiltration. Consequently, efficient separation of the DTTox from other flow-through mixture components (sugars, polymers, salts, protein stabilizers) was achieved on an Atlantis dC(18) column. After chromatographic separation, DTTox was selectively identified by UV absorbance at 285 nm or by selected reaction monitoring, measuring signal transition between m/z 151 -> 105. The method was validated in terms of specificity, accuracy, precision, linearity, and limit of quantification and detection. A reversed-phase HPLC separation method with atmospheric pressure chemical ionization and MS/MS detection in negative ion mode is highlighted as a viable alternative to currently existing quantitation methods involving DTT derivatization and HPLC fluorescence detection. The described approach offers simple, straightforward, selective, and high-throughput DTT quantitation in protein mixtures.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija, Farmacija

POVEZANOST RADA