Protein Footprinting via Covalent Protein Painting Reveals Structural Changes of the Proteome in Alzheimer's Disease

Abstract

Misfolding and aggregation of amyloid-β peptide and hyperphosphorylated tau are molecular markers of Alzheimer's disease (AD), and although the 3D structures of these aberrantly folded proteins have been visualized in exquisite detail, no method has been able to survey protein folding across the proteome in AD. Here, we present covalent protein painting (CPP), a mass spectrometry-based protein footprinting approach to quantify the accessibility of lysine ε-amines for covalent modification at the surface of natively folded proteins. We used CPP to survey the reactivity of 2645 lysine residues and therewith the structural proteome of HEK293T cells and found that reactivity increased upon mild heat shock. CPP revealed that the accessibility of lysine residues for covalent modification in tubulin-β (TUBB), in succinate dehydrogenase (SHDB), and in amyloid-β peptide (Aβ) is altered in human postmortem brain samples of patients with neurodegenerative diseases. The structural alterations of TUBB and SHDB in patients with AD, dementia with Lewy bodies (DLB), or both point to broader perturbations of the 3D proteome beyond Aβ and hyperphosphorylated tau.

Keywords: MudPIT; bottom-up proteomics; chemical footprinting; conformational diagnostics; diffuse Lewy body disease; isobaric isotopologue; molecular diagnostics; neurodegenerative diseases; protein surface mapping; quantitative mass spectrometry; structural proteomics.

Figure 1. Covalent Protein Painting (CPP) determines whether the ε-amino group of lysine is accessible for covalent modification.

(A) The schematic displays the workflow of CPP. Reductive alkylation labels lysine residues with isotope-defined “light” dimethyl moieties in living cells. Following digestion into peptides with a lysine-insensitive protease (Chymotrypsin), newly solvent exposed lysine residues are modified with isotope-defined “heavy” dimethyl moieties. Bottom up mass spectrometry is used to analyze the ratio of light to heavy isotope labeled peptide molecules per lysine site. (B) Lysine residue GAPDH#K309 is only partially accessible for chemical dimethylation in HEK293T cells. Proteins in HEK293T cells were covalently modified with CPP using isobaric isotopologue methyl moieties with ¹³CH₃ for light and CDH₂ for heavy, and the relative surface accessibility determined as described in (A). Numbers above the bars indicate the position of the lysine residue in GAPDH. The y-axis is the ratio of light to heavy fragment ion counts normalized to the total number of ion counts shown below each bar. A ratio of R = 1:1 (log₂(1) = 0) indicates that the lysine site was accessible for chemical modification in 50 % of protein molecules. Ion counts (IC) denotes the sum of fragment ion peaks that contributed to the quantification. (C) One dimer of the GAPDH homo-tetramer is displayed (PDB: 4wnc). Partial spheres (blue) highlight solvent accessible surface area (SASA) of each individual lysine ε-amine (grey spheres). Lysine residues that were assayed with CPP in (B) are highlighted in red. GAPDH#K309 resides within the contact surface of two GAPDH monomers in the GPADH dimer and is almost completely inaccessible for covalent modification. (D) The bar graph shows accessibility of lysine sites for covalent modification in highly purified, native GAPDH tetramers (orange) or heat denatured GAPDH (grey) or when the initial labeling step was omitted (blue). CPP results obtained for GAPDH#K309 are highlighted (red box). (E) Blue-native gel^® electrophoresis of purified GAPDH indicates structural stability of the GAPDH homo-tetramer following chemical dimethylation. GAPDH was pre-incubated with labeling reagents formaldehyde (FA), sodium cyanoborohydride (HD), and the quencher ammonium bicarbonate (QE). Bovine serum albumin (BSA, 66 kD) was included as molecular size indicator. Tetrameric GAPDH protein complexes migrated distinctively faster following CPP. Error bars are standard deviation (σ). Abbreviations: DiM, dimethyl moieties; Tet., homo-tetramers.

Figure 2. CPP quantified protein unfolding in HEK293T cells upon mild heat shock.

(A) The frequency plot and Gaussian fit (black) shows the number of peptides (n) in which a lysine site was accessible for covalent modification as measured by CPP (log₂ R) in the proteome of control HEK293T cells. Log₂ R values were binned by integer. The inset highlights the frequency distribution of lysine sites that are predominantly inaccessible for covalent modification with CPP. The relative number of protein molecules [%] in which lysine sites were accessible or conversely inaccessible to covalent modification is indicated above the bar graph. (B) The scatterplot compares the relative number of protein molecules in which a lysine residue was accessible for covalent modification in control versus heat shock treated HEK293T cells. “Accessible” and “inaccessible” on the scale bars indicate lysine sites that were measured as either completely accessible or inaccessible for covalent modification. Individual lysine residues differed by Δ > 2 in relative accessibility for covalent modification (pink). Significantly different lysine sites passed the discovery threshold of q < 0.01 (Benjamini-Hochberg corrected, red). The control lysine site CRBT1#K54 of exogenously added Chymotrypsin (green) and overall mean (blue) are shown. The diagonal denotes no change in relative covalent modification. Error bars are standard deviation (σ).