Pulse labeling HDX MS None amide deuteration level <0.5 at 10ms of folding time GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Protein refolding and pulsed HDX were carried out using a custom-made four-syringe continuous-flow setup with three sequential mixing steps. Syringe 1 was filled with an aqueous solution of 75 μM acid-unfolded aMb and 30 μM bradykinin in 10 mM HCl. Syringe 2 contained water with dilute ammonium hydroxide. Both syringes were connected to mixer M1 via fused silica capillaries. Refolding was initiated by mixing the contents of syringes 1 and 2 at M1 in a 1:1 volume ratio at a flow rate of 5 μl/min each, resulting in a pH jump from 2 to 10. The outlet of M1 was connected to a folding capillary. Depending on the folding time different capillary i.d. values and lengths were used (10 ms: i.d. 20 μm, l 5 mm; 100 ms: i.d. 50 μm, l 8.5 mm; 1 s: i.d. 75 μm, l 38 mm).

Pulse labeling HDX MS None amide deuteration level 0.5-0.75 at 10ms of folding time GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Protein refolding and pulsed HDX were carried out using a custom-made four-syringe continuous-flow setup with three sequential mixing steps. Syringe 1 was filled with an aqueous solution of 75 μM acid-unfolded aMb and 30 μM bradykinin in 10 mM HCl. Syringe 2 contained water with dilute ammonium hydroxide. Both syringes were connected to mixer M1 via fused silica capillaries. Refolding was initiated by mixing the contents of syringes 1 and 2 at M1 in a 1:1 volume ratio at a flow rate of 5 μl/min each, resulting in a pH jump from 2 to 10. The outlet of M1 was connected to a folding capillary. Depending on the folding time different capillary i.d. values and lengths were used (10 ms: i.d. 20 μm, l 5 mm; 100 ms: i.d. 50 μm, l 8.5 mm; 1 s: i.d. 75 μm, l 38 mm).

Native exchange MS None amide deuteration level < 0.25 GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Protein refolding and pulsed HDX were carried out using a custom-made four-syringe continuous-flow setup with three sequential mixing steps. Syringe 1 was filled with an aqueous solution of 75 μM acid-unfolded aMb and 30 μM bradykinin in 10 mM HCl. Syringe 2 contained water with dilute ammonium hydroxide. Both syringes were connected to mixer M1 via fused silica capillaries. Refolding was initiated by mixing the contents of syringes 1 and 2 at M1 in a 1:1 volume ratio at a flow rate of 5 μl/min each, resulting in a pH jump from 2 to 10. The outlet of M1 was connected to a folding capillary. Depending on the folding time different capillary i.d. values and lengths were used (10 ms: i.d. 20 μm, l 5 mm; 100 ms: i.d. 50 μm, l 8.5 mm; 1 s: i.d. 75 μm, l 38 mm). Exchange was measured after one hour folding time.

Native exchange MS None amide deuteration level 0.25-0.50 GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Protein refolding and pulsed HDX were carried out using a custom-made four-syringe continuous-flow setup with three sequential mixing steps. Syringe 1 was filled with an aqueous solution of 75 μM acid-unfolded aMb and 30 μM bradykinin in 10 mM HCl. Syringe 2 contained water with dilute ammonium hydroxide. Both syringes were connected to mixer M1 via fused silica capillaries. Refolding was initiated by mixing the contents of syringes 1 and 2 at M1 in a 1:1 volume ratio at a flow rate of 5 μl/min each, resulting in a pH jump from 2 to 10. The outlet of M1 was connected to a folding capillary. Depending on the folding time different capillary i.d. values and lengths were used (10 ms: i.d. 20 μm, l 5 mm; 100 ms: i.d. 50 μm, l 8.5 mm; 1 s: i.d. 75 μm, l 38 mm). Exchange was measured after one hour folding time.

Native exchange MS None amide deuteration level more than 0.50 GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Protein refolding and pulsed HDX were carried out using a custom-made four-syringe continuous-flow setup with three sequential mixing steps. Syringe 1 was filled with an aqueous solution of 75 μM acid-unfolded aMb and 30 μM bradykinin in 10 mM HCl. Syringe 2 contained water with dilute ammonium hydroxide. Both syringes were connected to mixer M1 via fused silica capillaries. Refolding was initiated by mixing the contents of syringes 1 and 2 at M1 in a 1:1 volume ratio at a flow rate of 5 μl/min each, resulting in a pH jump from 2 to 10. The outlet of M1 was connected to a folding capillary. Depending on the folding time different capillary i.d. values and lengths were used (10 ms: i.d. 20 μm, l 5 mm; 100 ms: i.d. 50 μm, l 8.5 mm; 1 s: i.d. 75 μm, l 38 mm). Exchange was measured after one hour folding time.