Supplementary Data
Figure 1: Distribution of criteria for automated removal of 97% of detected peak groups by the
default IDEOM filters. Of 27,727 peak groups (m/z and retention time pairs), 82 were assigned with
confident metabolite identifications based on authentic standards, 506 were putatively identified
based on m/z and retention time and 373 unidentified peak groups remained after noise filtering
and identification procedures. Full details of IDEOM filtering procedures are recorded in the IDEOM
documentation (http://mzmatch.sourceforge.net/ideom/Ideom_Documentation.pdf). Noise filter
parameters applied to this study are listed in supplementary table 1. Filtering annotations and
confidence scores for all individual peak groups are recorded in the ‘alldata’ worksheet of the IDEOM
Excel file for this study: [insert weblink here]. NOTE: the total number of peaks is slightly higher than
usually detected in untargeted metabolomics studies because ‘greedy’ peak-picking parameters
were used in an attempt to improve detection of small isotopomer peaks. Nevertheless, the noise
filters appeared to effectively remove the excess noise from the dataset for metabolite
identification.
Unidentified:
Probable MS
artefacts
7%
Contaminants or
Background
22%
Other Filtered (inc.
Rt & ppm)
2%
Small or
Variable
peaks
11%
Metabolites
3%
MS artefacts
27%
LC noise or
Peakpicking
artefacts
28%
Condifident
identifications
0.3%
Putative
identifications
2%
Unidentified
possible metabolites
1%
Table 1: Parameters applied for data processing with XCMS, mzMatch and IDEOM.
XCMS
Method:
ppm:
peak width (min):
peak width (max):
S/N threshold:
Prefilter (# points):
Prefilter (intensity):
Mzdiff:
normal mzXML
2
5 seconds
100 seconds
2
2
500
0.001
mzMatch
Mzmatch grouping RT window:
Mzmatch grouping m/z ppm:
0.5 min
5 ppm
Relative Std Dev (RSD) filter:
Noise filter (codadw):
Intensity filter (LOQ):
Minimum detections #
RT window for related peaks:
1
0.5
500
2
0.1 min
IDEOM
Relative Std Dev (RSD) filter:
Noise filter (codadw):
Intensity filter (LOQ):
Minimum detections #
RT window for related peaks:
RT for id of authentic standards:
RT for id for calculated RT:
PPM for mass identification:
PPM after re-calibration
Ignore related peaks before RT:
RT window for complex adducts:
RT window for Duplicatepeaks:
RT window for Shoulderpeaks:
Intensity ratio for Shoulderpeaks:
0.25
0.75
5000
3
0.2
10
45
5
3
0
0.5
1
2
5
STRICT
min
%
%
ppm
ppm
min
min
min
min
to 1
(for ratio
Minimum detectable intensity:
1000 calc)
0.05 Unpaired
Statistical P-value:
Preferred DB: TrypDB
tbr
File 1: (Suppfile1.xlsx) Excel spreadsheet summary of identified and putatively identified metabolites
in procyclic-form Trypanosoma brucei. 13C-labelled metabolites are annotated in column J. Detailed
peak intensities for each metabolite and isotopomer are listed in the ‘RAWisotopomerPEAKS’ sheet.
All data from this study, and full functionality for data analysis and visualisation, are available in the
IDEOM file (insert weblink here)
File 2: (isotopes_091013080512_neg.pdf) PDF file containing all mass traces and graphs for
isotopomers of putatively identified metabolites from negative mode MS ionisation.
File 3: (isotopes_142459070512_pos.pdf) PDF file containing all mass traces and graphs for
isotopomers of putatively identified metabolites from positive mode MS ionisation.