Combining ultrarapid mixing with photochemical oxidation to probe protein folding

We demonstrate a new method to study protein folding by combining fast photochemical oxidation of proteins (FPOP) with ultrarapid microfluidic mixing to observe kinetics on the microsecond time scale. Folding proteins pass through a focused UV laser beam, creating OH radicals that label the select protein side chains and are analyzed with mass spectrometry. As a proof of principle, we demonstrate this method with hen egg lysozyme that shows at least two kinetic phases before 1 ms, which are compared with those observed by Trp fluorescence. This method provides another, complementary probe of the early, complex steps of protein folding.