Microbial Profiling Facility - MPF Center for Microbial Biotechnology (CMB) Kristian Fog Nielsen Associate professor, Ph.D, Center for Microbial Biotechnology (CMB), headed by Prof. Jens Nielsen BioCentrum-DTU Technical University of Denmark Penicillium brevicompactum on YES

What I need to know Sample matrix Sample preparation ? matrix depended Amount of sample available ? (mg, gram ??) Special metabolites ? Target analysis (lower detection limits, lower standard deviation), matrix depended glycerol, trehalose, …… - please come with suggestions Screening / metabolomics way (interesting field more reviews than original papers) How many samples ? Development of sample preparation and analytical validation will probably be the most time consuming part

Which platforms can we currently offer Rugged screening method for LC-UV-HRMS (ESI - and ESI +) of secondary metabolites based on reversed phase separation (C 18, Phenyl, …), >650 reference standard, >10 years experience with ESI, 25 with LC-UV screening Several image analysis tools for whole LC-UV data matrices Tools for finding analogues of known metabolites Working on image analysis of LC-HRMS data and combine with LC-UV Direct infusion MS (DIMS) HR-MS and MS/MS Nano interface so we can spray for a very long time….. New algorithm for handling HR-MS data

Monoisotopic masses H C % 13 C % O N Cl % 37 Cl % C 16 H 28 N 3 O 2 masse Da C 15 H 24 N 3 O 3 masse Da

Conversion from continuum to centroid data m/z resolution = = 8027 FWHM 0.055

Rhizonin A Strong hepatoxin

Which platforms can we currently offer Most of the primary metabolites and household metabolites are highly polar and/or ionic No retention on RP phases HILIC experience (Poly LC, NH 2 …..,) sugars, … can be interfaced with MS Ion chromatography (Dionex systems), not directly interfacable to MS Organic acids, phosphate etc. (suppressor and CD) Sugars and amino acids, using PAD Dionex claims to have a desalting device for interfacing to MS !

Which platforms can we currently offer GC-MS platform MCF for organic acids and amino acids and some other metabolites (routine) Quenching of cells Hydrolysis of proteins, measuring amino acids as ECF and DMFDMA (routine are being developed further) Can be combined with 13 C labeled sugars as the C position in AA can be determined from the EI + spectra Sugars as the acetates Fatty acids and sterols have occasional been done

What do you want ? Please come with suggestions Tailor make an analytical program Test samples ! sample amount ?

Our “HTS” strategy and setup

A B C D E F G H HPLC fractionation sec. per well Evaporate solvent → Bioassay Known bioactives Unknown Bioactive => Quick dereplication Our “HTS” strategy and setup