ESI-FTMS

SEC-ESI-FTMS combines the size separation based technique of SEC with one of the most powerful mass spectrometric techniques of FTMS offering high mass accuracy (ppm), ultrahigh resolving power (>10(i) 6) and the capability to perform tandem mass spectrometry. The technique enables generation of oligomer elution profiles, which can be used for accurate calibration of standard SEC data. Coupling of SEC to ESI-MS is further described in ref. [710],... 

In this ESI-FTMS screening proof-of-concept experiment, a DCL was generated using the hydrazone exchange reaction from two hydrazide fragments (1 and 2) and five aldehyde fragments (A-E) (Scheme 7.2). 

Follow-up ESI-FTMS experiments in our laboratory have been effective with a 10-fold reduction in CA II concentration (from 30 to 3 xM, 4 xg protein/DCL based on a 50 xL reaction volume) while retaining the ability to detect protein-ligand noncovalent... 

Four cationic palladium intermediates in the Pd(0)-catalysed three-component cascade double addition-cyclization of organic halides, 2-(2,3-allenyl)malonates, and imines have been characterized by the high-resolution ESI-FTMS technology.84... 

The ESI/FTMS trace of tRNAphe with a zoom of the isotopic cluster for the 24 anion. Peaks at —15 and +15 Da are due to minor impurities. Reproduced (modified) from Little D.P., Thannhauser T.W. and McLafferty F.W., Proc. Natl. Acad. Sci USA, 92, 2318, 1995, with permission. 

The applied MS instrumentation for a substrate specificity assay depends on the size of the carrier domain construct whose active site is monitored. If multidomain constructs or T domain constructs >20 kDa are investigated, active site mapping is required and, therefore, ESI-FTMS instruments have to be applied because of their established active site mapping capabilities described above. If the monitored T domain constructs are freestanding and <20 kDa, no active site mapping is required and low-resolution MS instruments can be utilized for characterization of loaded substrates too. 

MS-based substrate identification assays of tailoring enzymes depend on whether the biosynthetic substrate is T domain bound or not. If the biosynthetic substrate is T domain bound, the predicted native substrate is loaded on the T domain and the mass change upon the tailoring reaction is detected by ESI—FTMS. If the biosynthetic substrate is non-T domain bound, conversion of the predicted native substrate by tailoring reaction can be detected by low-resolution MS. Biosynthetic substrate tolerance assays for tailoring enzymes are conducted in a one-assay-one-substrate approach like substrate identification assays but with alternative biosynthetic substrates. 

Figure 14 In vivo and in vitro substrate screening of loading module of microcystin biosynthesis, (a) Microcystin and Adda, (b) Loading protein McyG of microcystin synthetase, (c) In vitro and in vivo substrate screening assays of McyG AT. (d) Characterized substrates of McyG AT, ,Vo and McyG AT, vitm by ESI-FTMS (observed and calculated mass shifts from holo McyG AT active site).

Figure 16 Lipidation of daptomycin. (a) Role of DptE and DptF in daptomycin lipidation. DptE adenylates decanoic acid and tethers it on T domain DptF for insertion into daptomycin. (b) DptE substrate identification assay (observed and calculated mass shifts from holo DptF characterized by ESI-FTMS).

The recent studies of Hansen et al51 and Wittmann et al,109 revealed a new mechanism for lipidation of lipopeptide biosynthesis such as mycosubtilin or daptomycin biosynthesis by application of ESI-FTMS. Both papers describe that fatty acid incorporation is catalyzed by an A domain with fatty acid specificity in the loading module of the mycosubtilin NRPS (Figure 15) or by a preassembly line A and T domain in daptomycin biosynthesis (Figure 16). 

Gatto et al m characterized the mechanism of L-pipecolic acid formation by cyclodeaminase RapL from L-lysine within rapamycin biosynthesis, which is a hybrid NRP—polyketide antibiotic (Figure 25(a)). RapL was characterized by biochemical assays to require cofactor nicotinamide adenine dinucleotide (NAD+) and an oxidative cyclodeamination reaction mechanism corresponding to ornithine cyclodeamination was proposed based on ESI-FTMS analysis of RapL reaction products (Figure 25(b)). 

Figure 25 L-Pipecolic acid formation by cyclodeaminase RapL in rapamycin biosynthesis, (a) Rapamycin and incorporated pipecolic acid moiety, (b) Proposed oxidative cyclodeamination mechanism of pipecolic acid formation from L-lysine. (c) RapL activity assays and exact ESI-FTMS analysis of derivatized reaction products revealing mechanistic insights such as a-H retainment and loss of e-N.

Figure 26 Cyclopropane ring formation by CmaC in coronamic acid biosynthesis, (a) Characterization of 7-CI loss by ESI-FTMS. (b) Characterization of a-H exchange by deuterium solvent exchange and PEA. CmaC promotes deuterium incorporation at a-C position, (c) Proposed cyclopropane ring formation mechanism.55...

Figure 28 Investigation of interchain and intrachain acylation in dimeric VibF. (a) Vibrobactin. (b) VibF heterodimer for interchain and intrachain kinetic assay and ESI-FTMS analysis, (c) Obtained best-fit kinetic parameters for VibF intrachain and interchain acylation, Pinterchain - probability of interchain flux, /rinterohain - interchain acylation rate, /rintra0hain - intrachain acylation rate.

ESI-FTMS spectrometry electrospray ionization-Fourier transform mass spectrometry... 

---