N-terminal sequencing of protonated peptides is challenging, since each b2 ion represents two sequence isomers, e.g., NE and EN. Additionally the occurrence of compositional isomers, such as NE and QD, further increases the number of isomers to four (NE, EN, QD, DQ). This leads to a subset of 13 b2 ion masses where each value represents four individual species. The b2 ions within such a quartet are characterized by the same elemental composition. To test the utility of CID for differentiation of isomeric b2 ions, the CID spectra of 52 small synthetic peptides were recorded, representing the 13 isomeric b2 ion quartets, which may be formed from unmodified amino acid residues. The CID spectra of protonated peptides containing these quartets were carefully inspected for N-terminal sequence information. Below the m/z value of the b2 ion, individual differences were found in the b2 fragment ion signatures (neutral loss of CO, H2O, NH3, and other less common units). Recognition of N and Q in second position from the N-terminus is based on c1 ion formation. Relative intensities of immonium ions were also used for differentiation between sequence isomers. In the complementary high-mass regions above the m/z value of the ymax-2 ion, individual differences were observed in the formation of ymax-1, xmax-1 and zmax-1 ions, which could be correlated to the complementary low-mass ions. In summary, de novo sequencing of the N-terminal dipeptide motif is feasible by considering all available sequence information present in CID spectra of protonated peptides.