Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry

Abstract

Protein digestion is a key challenge in mass spectrometry (MS)-based structural proteomics. Although using hydrogen-deuterium exchange kinetics with MS (HDX-MS) to interrogate the high-order structure of proteins is now established, it can be challenging for β-barrel proteins, which are important in cellular transport. These proteins contain a continuous chain of H-bonds that impart stability, causing difficulty in digestion for bottom-up measurements. To overcome this impediment, we tested organic solvents as denaturants during on-line pepsin digestion of soluble β-barrel proteins. We selected green fluorescent protein (GFP), siderocalin (Scn), and retinol-binding protein 4 (RBP4) as model proteins and screened six different polar-aprotic and polar-protic solvent combinations to disrupt the H-bonds and hydrophobic interactions holding together the β-sheets. The use of organic solvents improves digestion, generating more peptides from the rigid β-barrel regions, without compromising the ability to predict the retinol binding site on RBP4 when adopting this proteolysis with HDX.

Figure 1.

Scan statistics for GFP under quench conditions with various organic solvents. The % coverage and number of unique peptides are annotated on the y axis.

Figure 2.

(a) An example of segment-length determination. The observed peptides are covered by black lines; the red dashed lines illustrate the ability to pinpoint residues or small sequences (segments) based on peptide overlap. Six segments are present in this example; their lengths are indicated. (b) Digestion coverage of representative peptic peptide 130–151 from GFP in the presence (green lines) and absence (black lines) of DMF.

Figure 3.

3D structure of a) GFP (PDB: 1GFL), b) Scn (PDB: 4K19), c) RBP4 (PDB: 5NU7). Sites that are cleaved more frequently in the presence of organic solvent (light pink) are located on beta-barrels peptides (light orange). Sites that are cleaved only in the presence of organic solvent are shown in light blue.

Figure 4.

Average percentage differences in deuterium uptake with MeOH addition between retinol-free and retinol-bound RBP4 mapped onto the X-ray crystal structure (PDB 5NU7) of retinol-bound RBP4. Insets: retinol-free (black) and retinol-bound RBP4 (dark-green).