Transcript Document

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
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Sample matrix
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Sample preparation ? matrix depended
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Amount of sample available ? (mg, gram ??)
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Special metabolites ?
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Target analysis (lower detection limits, lower standard deviation),
matrix depended
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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 (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
Monoisotopic masses
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H
12C
13C
O
N
35Cl
37Cl
1.0078
12.0000
13.0034
15.9949
14.0031
34.9689
36.9659
C16 H28 N3 O2 masse
C15 H24 N3 O3 masse
98.9%
1.1%
76%
24%
294.2182 Da
294.1818 Da
Conversion from continuum to centroid data
0.055 m/z
resolution =
444.231 = 8027
0.055
FWHM
O
Rhizonin A
Strong hepatoxin
O
N
N
N
O
O
O
N
N
O
N
N
O
O
O
kfn_cbs41 556 (12.375)
1: TOF MS ES+
812.4915
3.21e4
100
[M+H]+
Calc. 812.4922
[M+Na]+
Calc. 834.4741
834.4743
%
835.4753
339.2883
350.2081 463.2926 480.3106 502.2916
0
300
350
400
450
500
550
614.3648
600
699.4188
650
700
836.4857
727.4377
m/z
750
800
850
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
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Organic acids, phosphate etc. (suppressor and CD)
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Sugars and amino acids, using PAD
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Dionex claims to have a desalting device for interfacing to
MS !
Which platforms can we currently offer
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GC-MS platform
•MCF for organic acids and amino acids and some other
metabolites (routine)
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Quenching of cells
•Hydrolysis of proteins, measuring amino acids as ECF and
DMFDMA (routine are being developed further)
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Can be combined with 13C labeled sugars as the C
position in AA can be determined from the EI+ spectra
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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
Fraction collector
UV
mAU
Semi-Prep HPLC
60
50
40
30
Extraction of
0.5 cm2 to
whole plate
20
Ethyl acetate
10
0
200
Anti
bact
e
300
400
500
nm
rial
Analytical
LC-UV-HR-MS
ESI+ and ESI-
Anti-cancer
Daugther plates
With bioactive
wells
C18 / C8
al
fung
i
t
n
A
Master plate
1000
0
2.5
A
B
C
D
E
F
G
H
5
7.5
10
1 2 3 4 5 6 7 8 9 10 11 12
Evaporate solvent → Bioassay
12.5
15
17.5
P 1- F:05
P 1- F:06
P 1- F:07
P 1- F:08
P 1- F:09
P 1- F:10
P 1- F:11
P 1- F:12
P 1- F:03
P 1- F:04
P 1- E:10
P 1- E:11
P 1- E:12
P 1- F:01
P 1- F:02
P 1- E:08
P 1- E:09
P 1- D:12
P 1- E:01
P 1- E:02
P 1- E:03
P 1- E:04
P 1- E:05
P 1- E:06
P 1- E:07
P 1- D:03
P 1- D:04
P 1- D:05
P 1- D:06
P 1- D:07
P 1- D:08
P 1- D:09
P 1- D:10
P 1- D:11
P 1- D:01
P 1- D:02
P 1- C:07
P 1- C:08
P 1- C:09
P 1- C:10
P 1- C:11
P 1- C:12
P 1- B:11
P 1- B:12
P 1- C:01
P 1- C:02
P 1- C:03
P 1- C:04
P 1- C:05
P 1- C:06
P 1- B:02
P 1- B:03
P 1- B:04
P 1- B:05
P 1- B:06
P 1- B:07
P 1- B:08
P 1- B:09
P 1- B:10
2500
P 1- A:06
P 1- A:07
P 1- A:08
P 1- A:09
P 1- A:10
P 1- A:11
P 1- A:12
P 1- B:01
P 1- A:02
P 1- A:03
P 1- A:04
P 1- A:05
Our “HTS” strategy and setup
mAU
Known bioactives
2000
1500
500
0
20
Unknown Bioactive
HPLC fractionation
=> Quick dereplication
15-40 sec. per well
22.5
min