Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for (13)C-MFA

In recent years the benefit of measuring positionally resolved (13)C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of (13)C-Metabolic Flux Analysis ((13)C-MFA) has become evident. However, the usage of positional labeling information for (13)C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our (13)C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved (13)C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-016-0174-9) contains supplementary material, which is available to authorized users.