InSourcerer: a high-throughput method to search for unknown metabolite modifications by mass spectrometry

Rationale: Using mass spectrometry, the analysis of known metabolite structures has become feasible in a systematic high-throughput fashion. Nevertheless, the identification of previously unknown structures remains challenging, partially because many unidentified variants originate from known molecules that underwent unexpected modifications. Here, we present a method for the discovery of unknown metabolite modifications and conjugate metabolite isoforms in a high-throughput fashion. Methods: The method is based on user-controlled in-source fragmentation which is used to induce loss of weakly bound modifications. This is followed by the comparison of product ions from in-source fragmentation and collision-induced dissociation (CID). Diagonal MS2-MS3 matching allows the detection of unknown metabolite modifications, as well as substructure similarities. As the method relies heavily on the advantages of in-source fragmentation and its ability to 'magically' elucidate unknown modification, we have named it inSourcerer as a portmanteau of in-source and sorcerer. Results: The method was evaluated using a set of 15 different cytokinin standards. Product ions from in-source fragmentation and CID were compared. Hierarchical clustering revealed that good matches are due to the presence of common substructures. Plant leaf extract, spiked with a mix of all 15 standards, was used to demonstrate the method's ability to detect these standards in a complex mixture, as well as confidently identify compounds already present in the plant material. Conclusions: Here we present a method that incorporates a classic liquid chromatography/mass spectrometry (LC/MS) workflow with fragmentation models and computational algorithms. The assumptions upon which the concept of the method was built were shown to be valid and the method showed that in-source fragmentation can be used to pinpoint structural similarities and indicate the occurrence of a modification.