title: Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry.
abstract: The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one μs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 μs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 μs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 μs is obtained for the formation of a β-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 μs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 μs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 μs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720-1460, United States.
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Mortensen
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720-1460, United States.
authors.forename: Evan R
authors.initials: ER
authors.lastname: Williams
mesh_terms: D045304:Cytochromes c / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
chemical_list: D009211:Myoglobin; D010455:Peptides; D011506:Proteins; C508725:Trp-cage peptide; D045304:Cytochromes c