Microbial Profiling Facility - MPF Center for Microbial Biotechnology (CMB) Kristian Fog Nielsen Associate professor, Ph.D, Center for Microbial Biotechnology.

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Microbial Profiling Facility - MPF

Center for Microbial Biotechnology (CMB)

Kristian Fog NielsenAssociate professor, Ph.D,Center for Microbial Biotechnology (CMB), headed by Prof. Jens NielsenBioCentrum-DTUTechnical University of Denmark

Penicillium brevicompactum on YES

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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

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Which platforms can we currently offer

• Rugged screening method for LC-UV-HRMS (ESI- and ESI+) of secondary metabolites based on reversed phase separation (C18, 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

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Monoisotopic masses

• H 1.0078• 12C 12.0000 98.9%• 13C 13.0034 1.1%• O 15.9949• N 14.0031• 35Cl 34.9689 76%• 37Cl 36.9659 24%

C16 H28 N3 O2 masse 294.2182 Da

C15 H24 N3 O3 masse 294.1818 Da

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Conversion from continuum to centroid data

0.055 m/z

resolution = 444.231 = 8027 FWHM 0.055

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N

N

N

N

N

NN

O

O

O

O

O

O

O

O

ORhizonin AStrong hepatoxin

300 350 400 450 500 550 600 650 700 750 800 850m/z0

100

%

kfn_cbs41 556 (12.375) 1: TOF MS ES+ 3.21e4812.4915

339.2883480.3106350.2081 463.2926 502.2916 614.3648 699.4188

727.4377

834.4743

835.4753

836.4857

[M+Na]+

[M+H]+

Calc. 834.4741

Calc. 812.4922

300 350 400 450 500 550 600 650 700 750 800 850m/z0

100

%

kfn_cbs41 556 (12.375) 1: TOF MS ES+ 3.21e4812.4915

339.2883480.3106350.2081 463.2926 502.2916 614.3648 699.4188

727.4377

834.4743

835.4753

836.4857

[M+Na]+

[M+H]+

300 350 400 450 500 550 600 650 700 750 800 850m/z0

100

%

kfn_cbs41 556 (12.375) 1: TOF MS ES+ 3.21e4812.4915

339.2883480.3106350.2081 463.2926 502.2916 614.3648 699.4188

727.4377

834.4743

835.4753

836.4857

300 350 400 450 500 550 600 650 700 750 800 850m/z0

100

%

kfn_cbs41 556 (12.375) 1: TOF MS ES+ 3.21e4

300 350 400 450 500 550 600 650 700 750 800 850m/z0

100

%

kfn_cbs41 556 (12.375) 1: TOF MS ES+ 3.21e4812.4915

339.2883480.3106350.2081 463.2926 502.2916 614.3648 699.4188

727.4377

834.4743

835.4753

836.4857

[M+Na]+

[M+H]+

[M+Na]+

[M+H]+

Calc. 834.4741

Calc. 812.4922

Calc. 834.4741

Calc. 812.4922

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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, NH2…..,) 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 !

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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 13C 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

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What do you want ?

• Please come with suggestions

• Tailor make an analytical program

•Test samples !

• sample amount ?

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Our “HTS” strategy and setup

Extraction of0.5 cm2 to

whole plateSemi-Prep HPLC

nm 200 300 400 500

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Fraction collector

Antibacterial

Antifungal

C18 / C8

Daugther plates With bioactive

wells

Ethyl acetate

Anti-cancer

Master plate

AnalyticalLC-UV-HR-MSESI+ and ESI-

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ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12

HPLC fractionation15-40 sec. per well

Evaporate solvent → Bioassay

Known bioactives

Unknown Bioactive=> Quick dereplication

Our “HTS” strategy and setup