Erratum: Expression of N-terminal cysteine Aβ₄₂and conjugation to generate fluorescent and biotinylated Aβ₄₂ (Biochemistry (2021) 60:15 (1191-1200) DOI: 10.1021/acs.biochem.1c00105)

Gober et al. recently reported that maleimide conjugates of N-terminal cysteine peptides rearrange to the isomeric thiazines over the course of hours at neutral or basic pH.1 {equation presented}. To evaluate the occurrence of this rearrangement in the maleimide conjugates of Aβ(C1-42) reported in the original work,2 we have performed ultraperformance liquid chromatography. high-energy collision-induced mass spectrometry (UPLC.MSE) analysis of 5-TAMRA-Aβ(C1-42) (Figure 1). Upon digestion of 5-TAMRA-Aβ(C1-42) with proteinase K at 37 °C and pH 7.2 followed by UPLC-MSE, we were able to identify a set of three peaks in the UPLC trace associated with 5-TAMRA-Aβ(C1-7) (14.4, 16.3, and 16.5 min) and a set of three peaks in the UPLC trace associated with 5-TAMRAAβ(C1-5) (15.9, 17.8, and 18.1 min). The patterns of three unique peaks associated with each digestion fragment are consistent with the thiazine isomer and the two diastereomers of the maleimide conjugate, respectively. Similar patterns of three peaks were reported by Gober et al. for maleimide conjugates of several model tripeptides bearing N-terminal cysteine residues, with the thiazine isomer eluting first and the two diastereomers of the maleimide conjugate eluting subsequently.1 The observation of these characteristic sets of three peaks provides strong evidence that 5-TAMRAAβ(C1-42) has partially rearranged to the thiazine isomer in the course of preparation and isolation. We expect that 6- FAM-Aβ(C1-42) and PEG2-biotin-Aβ(C1-42) also partially rearranged to the thiazine isomers in the course of preparation and isolation. We further expect these Aβ(C1-42) maleimide conjugates to fully rearrange to the corresponding thiazine isomers during the course of common experiments that involve prolonged incubation at neutral pH, such as ThT fluorescence assays. Because N-terminal substitution does not substantially alter the properties of the Aβ, we anticipate that {figure presented}. the isomerization of the maleimide conjugates to the isomeric thiazines will not significantly affect the use of the Nterminally labeled Aβ42 peptides we have reported.