Small Molecule Discovery from LC-MS Data with Metaboseek

Maximilian J. Helf*, Bennett W. Fox, Frank C. Schroeder

Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
*Present address: Natural Products and Biomolecular Chemistry Unit, Novartis Institutes of BioMedical Research, Basel, Switzerland.
Contact: maximilian.helf@gmail.com

Data Import Feature Table Interactive Data Browser Structure Elucidation Peptide App Built From
Load feature tables
Navigation of data is centered on the feature table, a list
of molecular features, defined by at least m/z and retention
Browse, filter and annotate LC/MS features of interest.
Use powerful visualization tools to view and export
Use integrated tools to aid structure elucidation of molecular fea-
tures of interest. Save time by viewing the results in Metabo-
Metaboseek Modules
from other MS data External tools time (rt). Molecular features can be identified in MS data high-quality data plots. seek with quick access to the original raw data.
analysis tools or run
Import/ with Metaboseek’s xcms module or using external tools.
Customizable EIC Display Submit & View SIRIUS Jobs Metaboseek is built from individual modules that can be rear-
xcms analyses on xcms (Online) ranged easily into new shiny apps.
your MS data directly Plot extracted ion chromatograms (EICs) of molecular fea- Run SIRIUS³ analysis
from the Metaboseek MetaboAnalyst Export m/z rt {intensities, FC, p-value, ...} tures across files and arrange data in groups for rapid from the Metaboseek in-
interface.
mzMINE manual inspection terface and view results.
b2 b3 Use the PTModseek app Build a graphical user
LC/ HRMS 556.234 3.45 0 412 0 ... Export reports for individ- to annotate MS2 spectra interface for your data
Group 1 Group 2 ual molecular features, LL I
3.0e+07
of modified peptides analysis scripts
678.341 4.46 124 31 142 ... including their EIC and
SIRIUS fragmentation
XCMS 199.143 7.25 70 33 725 ... tree. Alternatively, open

227.1754+ ( 0.1)
340.2595+ ( 0.0)
500.2902+(−0.2)

658.3604+(−1.8)

873.4905+(−4.9)
990.5084+ ( 2.7)[1Me|1CAM]
1207.563+ ( 0.3)[1Me|3CAM]
1207.563+ ( 0.3)[1Me|2CAM]

1437.649+ ( 2.8)[1Me|2CAM]

1684.712+ ( 2.7)[1Me|3CAM]
1685.722+(−4.1)
786.453+ ( 1.5)
the analysis results in
Module
2.0e+07

MS Data Files Comparative Analysis SIRIUS GUI.

R
bn bn+1
O
Normalize feature intensities and run statisti- Label Finder N
H
N
N
cal analyses to compare sample groups for H H

[1CAM]

[1CAM]
[1CAM]
[1CAM]
Use the label finder module to detect stable
Parent O R
1.0e+07
feature-specific fold-change, t-test and y m+1 ym
isotope labeled compounds.
ANOVA. Import and export to MetaboAnalyst m/z b2 b3 b4 b6 b7 b8 b9 b10 b11 b13 b15 b16
b- and y- ions

Intensity

Relative Intensity
L L I C S A K S C C G E T S CWA C G
Accelerating Compound for advanced statistics. y5 y4 y3 y2

653.2164+ ( 0.9) [2CAM]

493.187+(−1.4) [1CAM]
307.1071+(−0.2) [1CAM]
236.0698+ ( 0.5) [1CAM]
Discovery C1
0
13
MS Data Reanalysis
11.8 12.0 12.2 12.4 11.8 12.0 12.2 12.4

13
C4
RT (min) RT (min)
13
C2 C3
13
C5
Comparative analysis of high resolution mass spectrometry (HRMS)
Reanalyze raw data for molecular features to
add information such as peak quality, exis-
MS/MS Browser 13
3.482e+04 RT: 4.496 min

y+2[1CAM]
100
View and compare mass spectra, including MS2 spectra Parent m/z: 1088.95398
data is a powerful approach to identify products and intermediates of

b+2
tence of custom fragmentation patterns MS2 m/z
belonging to individual nodes of a molecular network 80
biosynthetic pathways. However, making data analysis tools available data, or molecular formula database matches.
OH

Relative Intensity (%)

and moving between them is presenting a productivity bottleneck, 100
OH
572.2867 O 60
potentially leading to missed discovery opportunities. Custom Filters O

b+ +15[1Me|3CAM]
O

y+3[1CAM]

b+13[1Me|2CAM]
O O O

333.0607

b+11[1Me|2CAM]
HO

b+9[1Me|1CAM]
+ y4[1CAM]
P
40
N

b+8[1CAM]
Use results from comparative analysis, fea-

y++5[2CAM]
b6[1CAM]
b4[1CAM]
152.9965
To facilitate the identification of biologically relevant small molecules, HO

b+7[1CAM]
ture detection and MS data reanalysis to filter OH H

+
OH 20

336.1960

b+3
we recently introduced Metaboseek¹, an R/shiny-based, discovery-ori-

b16
for the features you want to investigate and
ented platform to extract differential features from HPLC-HRMS data
in a friendly graphical user interface. It allows you to rapidly inspect
add custom comments to molecular features. Feature-based Molecular Networking 0
500 1000 1500
410.2331 m/z
feature-specific data, find groups of structurally related com- Generate and browse molecular
0
networks based on MS2 similarity. Data shown here was previously published⁴ and reanalyzed with
pounds and assist compound identification and dereplication. Fold-change Peak Quality 100
Networks can also be exported to the PTModseek app, (included in the Metaboseek installer).
586.3027
Cytoscape. Visit Aileen Lee’s poster to learn about a new project using
Metaboseek features a convenient interface to run the xcms² pack-
*

333.0608
PTModseek in the Freeman lab.

Fold change
age for MS data processing and feature detection, and tools to filter for

152.9964
the most relevant molecular features using statistical analysis, predic-

350.2119
tion of molecular formulas, annotation of MS2 spectra, MS2 molecular
networking and finding of stable isotope labeled molecules.
0
424.2497
Get Metaboseek
100 200 300 400 500 Metaboseek is available as an installer for Win-
etabo dows, or as a cross-platform R package. You can
Summary Plots download the software and try an online preview
seek OH OH Aggregate data from the feature table into customizable version on our website at
plots for overview and easily create high quality figures.
MS2 Patterns Molecular Formulas
metaboseek.com
O
OH
m/z

Preliminary identifi- C H N O P S ... 350

+ cations for further OH

References Support
analysis R
300
OH
O 10
hacl-1 fold over WT
O
250 1. M. J. Helf, et al., Nat. Commun., 2022, 13, 1.
OH OH O
11:0 HO 2. C. A. Smith, et al., Anal. Chem., 2006, 78, 779.
10:0 N R 1 DFG Fellowship,
200
9:0 H Project #386228702
8:0 3. K. Dührkop, et al., Nat. Methods, 2019, 16, 299.
LC/ HRMS RT (sec) 600 800 1000
4. M. J. Helf, et al., ChemBioChem, 2017, 18, 444.
NIH U01