Fig. 1: Method development for intact protein MS.

Whole blood from a transgenic fALS mouse model (SOD1^G93A) fractionated using chloroform-ethanol without (a–e) and with (b–f) spiked S-XL6 (200 µM). S-XL6 is a kinetic stabilizer that cross-links the SOD1 dimer via two cysteines (Cys111_{subunit A} and Cys111_{subunit B}). Cross-linking forms two disulfide bonds that may be susceptible to cleavage by thiol-disulfide exchange with endogenous thiols (e.g., glutathione)⁷⁹. To address this, we compared target engagement without (a, b, green) and with (c, d, purple; e, f, light blue) thiol alkylating (i.e., endogenous thiol blocking) agents iodoacetamide (IAA) and N-ethyl maleimide (NEM). The inset panels show the deconvoluted spectra. Ubiquitin peak eluted at 23.5 min. Alkylating agents did not improve cross-linking yield, indicating that a thiol blocking step was unnecessary in the final method. Mass spectra were obtained using reversed-phase liquid chromatography and quadrupole time-of-flight MS (Agilent 6560 LC-QToF-MS). % TE is defined as percentage of deconvoluted intensity of the drug-protein complex [drug-protein complex intensity / (drug-protein complex intensity + unbound protein intensity)]. Total ion current is represented as a.u. (arbitrary units) and mass is reported in kDa.