Mass Defect Filtering: a New Tool to Expedite Screening, Dereplication and Identification of Natural Products
Li-Quan Wang and Zhe-ming Gu
XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, NJ 08536
52.8
-25
-9.4
Kaempferol
Quercetin
Myricetin
Isorhamnetin
Pachypodol
Rhamnazin
Hesperetin
Naringenin
Eriodictyol
Homoeriodictyol
O
OH
O
Flavonol
O
O
Flavanone
C15H10O6
C15H10O7
C15H10O8
C16H12O7
C18H16O7
C17H14O7
C16H14O6
C15H12O5
C16H12O6
C16H14O6
286
302
318
316
344
330
302
272
288
302
47.7
42.7
37.6
58.3
89.6
74.0
79.0
68.5
63.4
79.0
-9
7
23
21
49
35
7
-23
-7
7
-14.5
-19.5
-24.6
-3.9
27.4
11.8
16.8
6.3
1.2
16.8
O
Taxifolin
OH
C15H12O7
304
58.3
-3.9
9
O
Flavanonol
Genistein
Daidzein
Glycitein
O
O
Isoflavone
C15H10O5
C15H10O4
C16H12O5
270
254
284
52.8
57.9
68.5
-25
-41
-11
-9.4
-4.3
6.3
O
Catechin
C15H14O6
290
79.0
-5
16.8
Flavan
Average
295
62.2
Table 1. Mass and mass defect values for common flavanoids.
Results and Discussion
1. Principles of MDF in Screening Natural
Products
Many natural products exist as isomers or analogues,
and the mass defects of the same series of natural
products are similar, i.e. the different flavonoids as
shown in Table 1.
(E+7)
5.4
A. TIC before filtering
15.58
51.49
48.53
57.18
4.5
Intensity
70.23
3.6
68.00
2.7
0
(E+6)
46.88
B. TIC after filtering
47.98
Thermo LTQ Orbitrap XL
4.5 kV
350 °C
60
40
60
185.16479
440.10421
40
20
687.50042
668.28171
231.17037
314.13873
0
200
250
300
382.21019
350
2.0
528.33886
400
450
444.31043
100
50.33
52.05
52.93
1.0
0.5
MS Systems:
Ion spray (IS):
Capillary temp:
Sheath gas:
Auxiliary gas:
A
80
500
550
600
650
700
B
80
600
650
700
10
20
30
40
Time (min)
50
60
70
Figure 1. Total ion chromatograms of the full scan data
comparing the unfiltered chromatogram (A) to the mass defect
filtered chromatogram (B).
460.30542
60
462.32112
40
20
0
150
0
446.32611
200
250
300
350
400
450
500
550
m/z
Figure 2. Mass spectra of minor alkaloids with m/z 444, m/z
446, m/z 460, and m/z 462 in the extract of Fritillaria
thunbergii obtained by LC/MS without (A) and with (B) MDF
processing.
N
OH
60
m/z 414
40
-H2O
HO
432.34688
C 27 H 46 O 3 N
-0.78641 ppm
OH
Peimine (MH+ 432 Da)
20
0
150
To screen different types of compounds, various MDF
templates should be designed.
For a series of natural analogues, the compound with
mass defect and nominal mass values close to the
respective mean mass defect and nominal mass values
should be selected.
200
250
300
350
400
m/z
426.33710
C 28 H 44 O 2 N
-1.03451 ppm
F: FTMS + c ESI d Full ms2 [email protected] [110.00[110.00 455.00]
-
B
100
80
138.12746
C9H16 N
-1.95327 ppm
N
140.14315
C9H 18 N
-1.6296 ppm
HO
H+
HO
+
O Puqienine B (MH 444 Da)
60
N
40
N
N
20
HO
m/z 140
O
444.34686
C 28 H 46 O 3 N
-0.80459 ppm
m/z 138
m/z 426
0
150
200
250
300
350
400
450
m/z
Figure 4. Product ion spectra of (A) m/z 432 (C27H46O3N) and
(B) m/z 444 (C28H46O3N) from Fritillaria thunbergii.
65.80
(E+8)
1.0
A. TIC before filtering
0.8
0.6
50.01
0.4
4. Rapid Partial Identification of Natural
Products
65.13
64.24
60.88
59.24
45.76
39.92
0.2
0
(E+6)
5.0
As shown in Figure 4, an alkaloid with a protonated
formula of C28H46O3N (m/z 444) can be easily
identified as a veratraman-type alkaloid based on the
comparison of its product ion spectrum with that of
Peimine (m/z 432), acquired by data dependent FTMS2 scan.
48.28
B. TIC after filtering using
glycyrrhizin as a template
4.0
47.49
48.69
51.10
3.0
47.23
2.0
44.59
0
(E+7)
2.5
59.76
C. TIC after filtering using
glycyrol as a template
2.0
61.91
62.46
1.5
57.60
57.19
1.0
64.62
54.71
0.5
0
0
10
20
30
40
Time (min)
50
60
70
Figure 3. Total ion chromatograms of the full scan data
comparing the unfiltered chromatogram (A) to the mass defect
filtered chromatograms using glycyrrhizin as a template (B)
and glycyrol as a template (C).
3. Application in Natural Product
Screening and Dereplication
611.37596
m/z
1.5
Natural source organisms typically contain many
novel and structurally diverse chemical types.
344.25421
150
Relative Abundance
Mass Spectrometry:
0.9
Intensity
Shimadzu LC-20AT Pumps
A: 10 mM ammonium acetate pH 5.6
B: MeOH
The protonated ions at m/z 444, m/z 446, m/z 460, and
m/z 462 were obscured by many matrix or surrounding
interference ions in the unprocessed full-scan MS data
(Figure 2A). Figure 2B depicts the same mass
spectrum processed with mass defect filtering. After
MDF processing, the majority of the interference ions
were removed and the protonated ions at m/z 444, m/z
446, m/z 460, and m/z 462 became predominant in the
mass spectrum.
A
80
1.0
100
1.8
Pump:
Mobile Phases:
Figure 1 illustrates the effectiveness of the MDF
(MetWorks 1.2) using Peimine as a filter template for
detecting alkaloids in the crude extract of Fritillaria
thunbergii. As shown in Figure 1A, a very large
number of ions were widely spread from 10 to 70 min.
The ion signals of the alkaloid analogues were buried
in the TIC before MDF processing. However, when an
MDF was applied to the whole LC-MS data set, the
resultant TIC was a remarkable contrast (Figure 1B), in
which the alkaloid analogues were easily detected in
the much cleaner and simplified ion chromatogram.
2. Strategy of Selecting MDF Template
100
Relative Abundance
270
C15H10O5
Flavone
Experimental
Liquid chromatography:
286
∆MD from
average
-14.5
Relative Abundance
Apigenin
O
C15H10O6
∆MW from
average
-9
Intensity
Conventional screening for bioactive natural products
in drug discovery involves bioassay-directed isolation
and purification, which is time-consuming and
expensive, and requires large amounts of crude plant or
microorganism extracts. The use of LC/MS has played
a valuable role in the screening of natural products as
part of drug development programs over the past
decade, facilitating rapid confirmation of known
compounds, dereplication of known active compounds,
rapid discovery of new compounds, etc. Our group
successfully applied LC/MS in natural product
screening for acetogenins using atmospheric pressure
in-source
collision-induced
dissociated
(CID)
techniques over a decade ago. Recently, the technique
of Mass Defect Filtering (MDF) has revolutionized the
drug impurity and metabolite identification by LC-MS,
and thus is being widely used in drug development.
This new technology has been demonstrated applicable
to natural product screening since analogue natural
products also have similar mass defects. The objective
of this study was to apply the MDF technology in
combination with high resolution LC/MS and MS/MS
in natural product chemistry to expedite the screening,
dereplication and identification of natural products.
MW (Da)
414.33621
C 27 H 44 O 2 N
-1.08537 ppm
F: FTMS + c ESI d Full ms2 [email protected] [105.00[105.00 445.00]
-
Intensity
O
Formula
With a mass defect window set approximately ±?0 mDa
around the mass defect of an applied filter template
over a mass range of ±?0 Da around the mass of the
filter template, the majority of interference ions falling
outside the specified range will be automatically
removed, and the resultant simplified data could
facilitate the screening of natural products.
Intensity
Group
Mass defect
(mDa)
47.7
Relative Abundance
Introduction
Compound
Name
Luteolin
Figure 3 shows the effectiveness of MDF for detecting
disaccharide conjugates in the crude extract of Radix
Glycyrrhizae.
The unprocessed base peak
chromatogram showed no distinct peaks (Figure 3A).
After MDF processing, the disaccharide conjugates
were easily detected in the ion chromatogram from ca.
40 to 55 min region (Figure 3B); and the coumarins or
flavonoids were easily detected in the ion
chromatogram after ca. 55 min region (Figure 3C).
For the compound with m/z 444, two fragment ions at
m/z 138 and m/z 140 were observed, consistent with
typical dissociation from veratraman-type alkaloids.
The fragmentation pathway of Puqienine B is shown
in Figure 4, but this compound is not necessarily
Puqienine B solely based on the LC/MS/MS data.
Conclusion
MDF has been demonstrated to be a powerful tool in
the natural product screening, dereplication and rapid
identification. Different type of natural products can
be screened with the post-acquisition MDF
processing using various templates. The known
compounds can be easily dereplicated and the new
compounds can be easily identified or partially
identified. The MDF processing may be also useful
in Traditional Chinese Medicine fingerprint and
processing.