Table 1. Combined List of HSA–Secosterol Adduction Sites Analyzed by High Resolution FTMS^a.

sequence	sites	precursor, m/z	charge	mass error, ppm
1DAHK*SEVAHR	K4	517.9285	3	6.5
187DEGK*ASSAK	K190	647.8977	2	1.1
182LDELRDEGK*ASSAK	K190	641.0491	3	3.7
191ASSAK*QR	K195	575.3854	2	2.7
191ASSAK&QR	K195	566.3806	2	3.6
196QRLK*C#ASLQK	K199	817.5283/545.3542	2/3	3.4/2.7
198LK*C#ASLQK	K199	675.4467/450.6332	2/3	1.6/0.9
198LK&C#ASLQK	K199	444.6304	3	2.5
414K*VPQVSTPTLVEVSR	K414	681.4367	3	4.0
433VGSK*C#C#K	K436	620.8777	2	–5.5
525K*QTALVELVK	K525	766.0295	2	2.3
525KQTALVELVK*HKPK	K534	506.0964	4	6.8

^aHSA was treated with either seco A or seco B followed by NaBH₄ reduction. SPE fractionation was performed Supporting Information Scheme 1). The majority of unmodified peptides were contained in the 30% MeCN fraction (Supporting Information Table 1). All the identified adducted peptides were found in the 60% MeCN fraction. Based on MS/MS fragmentation patterns, the final form of secosterol was identified as seco B or its dehydrated products. A separate list of adducted peptides is shown in Supporting Information Table 2. The same sites have been modified by both electrophiles repeatedly. Identified peptides were further confirmed by DDNL MS³. The mass accuracy and peptide fragmentation assignments are in good agreement with their covalent modification and sites. K* represents a seco B imine adduct on lysine. K^& is a dehydrated adduct on lysine. C^# is a carbamidomethyl modification on cysteine.