TY - JOUR

T1 - Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow

AU - Trabjerg, Esben

AU - Jakobsen, Rasmus Uffe

AU - Mysling, Simon

AU - Christensen, Søren

AU - Jørgensen, Thomas J D

AU - Rand, Kasper D

PY - 2015/8/6

Y1 - 2015/8/6

N2 - Analysis of disulfide-bonded proteins by HDX-MS requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench solution (e.g. TCEP). The chemical reduction, however, is severely limited under quenched conditions due to a narrow time window as well as low pH and temperature. Here, we demonstrate the real-world applicability of integrating electrochemical reduction into an online HDX-MS workflow. We have optimized the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and Nerve Growth Factor-β (NGF). Several different parameters (flow rate, applied square wave potential as well as the type of labeling- and quench buffer) were investigated, and the optimized workflow increased the sequence coverage of NGF from 46% with chemical reduction to 99%, when electrochemical reduction was applied. Additionally, the optimized workflow also enabled a similar high sequence coverage of 96% and 87% for the heavy and light chain of the IgG1-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction is poised to decrease the complexity of sample handling and increase the versatility of the HDX-MS technique.

AB - Analysis of disulfide-bonded proteins by HDX-MS requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench solution (e.g. TCEP). The chemical reduction, however, is severely limited under quenched conditions due to a narrow time window as well as low pH and temperature. Here, we demonstrate the real-world applicability of integrating electrochemical reduction into an online HDX-MS workflow. We have optimized the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and Nerve Growth Factor-β (NGF). Several different parameters (flow rate, applied square wave potential as well as the type of labeling- and quench buffer) were investigated, and the optimized workflow increased the sequence coverage of NGF from 46% with chemical reduction to 99%, when electrochemical reduction was applied. Additionally, the optimized workflow also enabled a similar high sequence coverage of 96% and 87% for the heavy and light chain of the IgG1-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction is poised to decrease the complexity of sample handling and increase the versatility of the HDX-MS technique.

U2 - 10.1021/acs.analchem.5b01996

DO - 10.1021/acs.analchem.5b01996

M3 - Journal article

C2 - 26249042

VL - 87

SP - 8880

EP - 8888

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 17

ER -