Prominent MS/MS ions of m/z 112 and 140, observed for 2, 5 and 11 and corresponding to Ada-Pro fragments were shifted by 14.015 a.m.u. (CH2) in compound 8. Given the absence of methyltransferases in the mic BGC of M. aeruginosa LEGE 91341, we propose that methyl proline (mPro) is present in 7, 8 and 9. Several cyanobacterial natural products of NRPS origin contain 4-mPro and the enzymes involved in the production of this non-proteinogenic amino acid have been characterized. We looked for close homologs of such enzymes (NosE, accession AAF17283. 91341 but could only find a homolog of the Δ 1 -pyrroline-5-carboxylic acid reductase NosF (39% identity and 55% similarity). Because these data correspond to a draft genome of M. aeruginosa LEGE 91341, it is possible that the strain does contain the enzymatic machinery necessary to generate 4-mPro. However, we refrain from ascribing the methylation to a specific Pro carbon. The MS/MS spectrum for compound 5 shows a prominent m/z 462 peak that is not observed in 2, while the m/z 478 peak corresponding to the neutral loss of Pro-Tyr in 2 is not observed in 5. The difference between these two fragments (15.994 a.m.u.) correspond to an oxygen atom. Considering that, as described above, the [Cl-Ada] and Ada-Pro fragmentations in 2 and 5 are identical, we ascribe the oxygen atom difference to a Hphe residue being present in 4, 5 and 6, instead of the Htyr residue found in 1, 2 and 3. Analogues of cyanobacterial secondary metabolites with this type of modification have been described previously and are related to the relaxed specificity of the corresponding NRPS adenylation domain. Overall, our findings confirmed that M. aeruginosa LEGE 91341 produces 12 new microginins. These congeners contain the less common Ada fatty acyl moiety and a new combination of amino acids when compared to previously reported microginins. In addition, neither Hphe nor mPro had previously been encountered in this large group of cyanobacterial peptides.