Figure 4

Mass spectra of A) albumin and B) TTR extracted from human plasma that was treated with/without hydrogen peroxide and with/without the Cys-SOH trapping reagent TNB. For albumin (A), deconvoluted "MH⁺" peaks are assigned as follows: 66439 = Native/unmodified (Calc. 66439), 66558 = S-Cysteinylated (Calc. 66558), 66602 = Glycated native form (Calc. 66601), 66636 = TNB-adducted native form (Calc. 66636), 66720 = Glycated S-Cysteinylated form (Calc. 66720). For TTR (B), 13762 = Native/unmodified (Calc. 13762), 13794 = Cys-sulfinic acid (SO₂H) due to over oxidation (Calc. 13794), 13881 = S-Cysteinylated (Calc. 13881), 13938 = S-CysGly (Calc. 13938), 13959 = TNB-adducted native form (Calc. 13959). Assigned "MH⁺" values in deconvoluted spectra are centroided average masses. All samples were immediately purified with a gel filtration spin column upon addition of TNB and further prepared as quickly as possible for introduction into the mass spectrometer (~ 3 minutes for diluted samples and <15 minutes for MSIA samples). Albumin was prepared by dilution and TTR was prepared by MSIA (with the exception of the - H₂O₂/⁺ TNB sample for TTR which was prepared by dilution to facilitate the fastest possible introduction into the mass spectrometer). Panels C and D provide extended views of the samples to which TNB was added, demonstrating that TNB reacts only with the form of TTR which originally carried a free thiol. As illustrated, TNB does not bind noncovalently to S-cysteinylated or "thiol blocked" TTR.