The pseudo-tetrapeptide designated here as RGD (N-ethyl-N-[1-oxo-4-(4-piperidinyl) butyl] glycyl-L-α-aspartyl-3-cyclohexyl-L-alaninamide) and its isomer with β-aspartic acid rather than α-aspartic acid were examined using electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). RGD has potential as a thrombosis inhibitor and the isomer, designated here as isopeptide, is an inactive instability product; hence, means were sought to distinguish the two. Both isomers give a protonated parent on ESI and fragments typical of peptides on sustained off resonance irradiation collision-induced decomposition (SORI-CID). Cleavage at the aspartic acid (b3) is the dominant process in both isomers, although a significant b2 and smaller a2 “and c2” peaks are also observed. More distinctive are peaks observed at b3 – H2O, b3 – (CO + CO2) and, only in the case of the RGD, b3 – (H2O + CO). SORI CID on the b3 ion indicates that, of these distinctive peaks, only the b3 – (CO + CO2) comes from decomposition of the b3 ion. On this basis, a mechanism is suggested for b3 formation, involving proton transfer from a back-bone carbonyl to the aspartic acid side-chain carboxyl group. Such an intramolecular proton transfer involves rings of different sizes for the two isomers, providing a basis for the different SORI energy dependences. A mechanism suggested for the formation of the b3 – H2O fragments also involves proton transfer to the aspartic acid side chain carboxyl group. This leads to concomitant H2O loss and amide bond cleavage, giving the b3 – H2O ions with ketene moieties resulting from the water loss. According to the suggested mechanism, the observed loss of CO (verified by SORI-CID on the b3 – H2O ion) from the RGD b3 – H2O peak results in a secondary carbocation stabilized by an adjacent nitrogen. The unobserved loss of CO from the b3 – H2O ion, formed by the suggested mechanism from the isopeptide, would give an unstable primary carbocation lacking a neighboring nitrogen. The mechanism, thus, only rationalizes the observation of a b3 – (H2O + CO) fragment in RGD and not in the isopeptide. The isomers can be distinguished on the basis of this unique peak or on the basis of the different SORI energy dependence of the formation of the b3 ions.