Ty of characteristic b2 ions formed during experiments. The b2 ion was identified for peptides containing Asp residue and the following fixed-charge tags: TEA, TPP, and TMPP. The proposed mechanism of formation with the b2 ion for derivatized peptides, which has been currently presented by Gu et al. [4], is shown in Scheme 3. Within the case of analogues derivatized with ABCO, the b2 ion was only observed for the -AHPC-amido-C5-acid PROTAC HGDGATL-NH2 (Figure S40) sequence in which no arginine or lysine residue is present. The fragmentation spectra of peptides derivatized using the two,four,6-triphenylpyridinium group (Figures S48, S52, S54 and S56) revealed the low-intensity signal corresponding for the b2 ion at m/z 463.151, proving slight impact of aspartic acid. Inside the case of peptides modified by the TMPP group, the b2 ion (m/z 688.212) is dominant within the spectra, that are shown in Figures S62, S68 and S70. Such an observation makes it possible for us to conclude unequivocally that the type of fixed-charge tag utilised has an influence around the presence with the aspartic acid impact. In addition, a significant influence with the aspartic acid effect was observed for the modified peptides with out standard amino acids in their sequence mainly because the salt bridges are not formed and dissociation just after aspartic acid can take place. Additional analysis of your aspartic acid impact was performed on peptides containing Ala residue as opposed to Asp at position two. It can be expected that the lack of aspartic acid within this position should inhibit the formation in the b2 fragment that was observed for AGRTL derivatized with TEA, ABCO, and TPP groups (Figures S22, S36 and S50). In the case of fragmentation on the TMPP -CH2 CO-AGRTL-NH2 derivative (Figure S64) beside the intensive a2 ion, the signal at m/z 644.221 corresponding for the b2 ion is observed. It is probably that the signal corresponding to this ion benefits in the preferences of the quaternary phosphonium group, and so that you can unambiguously explain this, another series of analogues containing other amino acid residues at position 2 needs to be tested. Lately, we analyzed the fragmentation pathways on the model peptide derivatized by 5-azoniaspiro[4.4]nonyl or benzo-5-azoniaspiro[4.4]nonyl ionization enhancers in the N-terminus [35,36]. The obtained benefits indicate a comparable tendency within the ESI-MS/MS spectra recorded for peptide derivatives within the form of ASN -COVESYVPLFP-OH and BASN -CO-VESYVPLFP-OH. Each and every from the analyzed N1-Methylpseudouridine custom synthesis azoniaspiro peptide derivatives made an intense signal corresponding to the a2 and b2 ions, which might be utilised as characteristic ions in LC-ESI-MRM evaluation. For that reason, it may be assumed that the fragmentation pathways are not predictable and depend on several elements, which make their detailed evaluation significant. two.3. ECD Evaluation Through the ECD experiment, the charge of a peptide ion is reduced as a result of electron transfer. Therefore, only multiple-charged (z two) molecules could possibly be analyzed by this technique [37]. In our study, the ECD approach was utilized to check the effect in the introduced fixed-charge tag on the fragmentation pattern. The product ions identified in ECD fragmentation from the studied peptides (3a , 7ad) are shown in Figure 11, Figure 12, and Figures S71 86. Throughout the analysis, one limitation was the somewhat low m/z worth (below 350 for charge two) and low intensity of your [MH]2 ion corresponding to peptides with TEA and ABCO groups, which impeded fragmentation (Figure 10 and Figures S71 78). On the other hand, an intensive signal corr.