Cell line dependence of metabolite leakage in metabolome analyses of
adherent normal and cancer cell lines
Journal: Metabolomics
Rahul Vijay Kapoore · Rachael Coyle · Carolyn A Staton · Nicola J Brown ·
Seetharaman Vaidyanathan
Rahul Vijay Kapoore · Seetharaman Vaidyanathan
ChELSI Institute, Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering,
The University of Sheffield, Mappin Street, Sheffield, S1 3JD, United Kingdom
Corresponding author email: s.vaidyanathan@sheffield.ac.uk
Ph.: +44 (0)114 222 7526
Fax: +44 (0)114 222 7501
Rachael Coyle · Carolyn A Staton · Nicola J Brown
Microcirculation Research Group, Department of Oncology, Faculty of Medicine, Dentistry and Health, The
University of Sheffield, Sheffield, S10 2RX, United Kingdom
Table S1 List of metabolites identified by GC-MS along with their retention time from twelve metabolite
mixture used for validation of AMDIS parameters
Number
1
2
3
4
5
6
7
8
9
10
11
12
Metabolite standards (50 µM each)
Citric acid (4TMS)
Glucose methoxyamine (5TMS)
L-Aspartic acid (3TMS)
L-Glutamic acid (3TMS)
L-Glutamine (3TMS)
L-Lysine (4TMS)
L-Methionine (2TMS)
L-Phenylalanine (2TMS)
L-Threonine (3TMS)
Malic acid (3TMS)
Putrescine (4TMS)
Arginine
Retention time
25.0271
26.1179
19.475
21.4956
18.6826
26.7507
19.5474
21.7003
16.4284
18.8048
17.3496
Not detected
Fig. S1 Overlay of total ion chromatograms after retention time correction using XCMS online (web-based
platform) of all trypsinized and cell scraped samples from MDA-MB-436 cell line. X-axis represents total
ion current (TIC) and Y-axis represents retention time in minutes, where Tryp = Trypsinization (straight
lines); Csr = Cell scraping treatment (dotted lines); 436 = MDA-MB-436 cell line and A, B and C =
biological replicates.
Fig. S2 Overlay of total ion chromatograms after retention time correction using XCMS online (web-based
platform) of all trypsinized and cell scraped samples from HMEC-1 cell line. X-axis represents total ion current
(TIC) and Y-axis represents retention time in minutes, where Tryp = Trypsinization (straight lines); Csr = Cell
scraping treatment (dotted lines); Hmec = HMEC-1 cell line and A, B and C = biological replicates.
Fig. S3 Overlay of total ion chromatograms after retention time correction using XCMS online (web-based
platform) of all trypsinized and cell scraped samples from MCF-7 cell line. X-axis represents total ion current
(TIC) and Y-axis represents retention time in minutes, where Tryp = Trypsinization (straight lines); Csr = Cell
scraping treatment (dotted lines); Mcf7 = MCF-7 cell line and A, B and C = biological replicates.
Fig. S4 PCA analysis of metabolomics data displaying clustering of MDA-MB-436 samples after trypsinization
and cell scraping treatment. PCA is calculated using the feature intensities from all samples. The colours
(red/blue) are assigned based on the sample class, where red colour = trypsinized samples; blue colour = cell
scraped samples; Tryp = Trypsinization; Csr = Cell scraping treatment; MDA = MDA-MB-436 cell line and A,
B and C = biological replicates.
Fig. S5 PCA analysis of metabolomics data displaying clustering of HMEC-1 samples after trypsinization and
cell scraping treatment. PCA is calculated using the feature intensities from all samples. The colours (red/blue)
are assigned based on the sample class, where red colour = trypsinized samples; blue colour = cell scraped
samples; Tryp = Trypsinization; Csr = Cell scraping treatment; Hmec = HMEC-1 cell line and A, B and C =
biological replicates.
Fig. S6 PCA analysis of metabolomics data displaying clustering of MCF-7 samples after trypsinization and cell
scraping treatment. PCA is calculated using the feature intensities from all samples. The colours (red/blue) are
assigned based on the sample class, where red colour = trypsinized samples; blue colour = cell scraped samples;
Tryp = Trypsinization; Csr = Cell scraping treatment; Mcf7 = MCF-7 cell line and A, B and C = biological
replicates.
Table S2 List of putatively identified metabolites in MDA-MB-436, HMEC-1 and MCF-7 cell lines across
different applied sampling protocols
Acetoacetic acid
Acetyl-L-serine
Aconitic acid
Adenine
Adipic acid
Alanine
Allose
Altrose
Aminocyclopropanecarboxylic acid
Apigenin
Arabinose
Arabitol
Arginine
Asparagine
Aspartic acid
beta-Alanine
Biotin
Butanoic acid
Butyric acid
Calystegine B2
Cholesterol
Cinnamic acid
Citramalic acid
Citric acid
Cysteine
Cytosine
Decanoic acid
Diethyleneglycol
Dihydroorotic acid
Dodecanoic acid
Eicosanoic acid
Eicosanol
Epinephrine
Erythritol
Erythronic acid
Erythrose
Ethanolamine
Ethylglucopyranoside
Flavone
Fructose
Fructose-6-phosphate
Fumaric acid
Galactinol
Galactonic acid
Galactosamine
Galactose
Galactose-6-phosphate
Galacturonic acid
Gentiobiose
Glucaric acid
Glucoheptonic acid-1,4-lactone
Gluconic acid
Gluconic acid-1,5-lactone
Gluconic acid-6-phosphate
Glucose
Glucose-6-phosphate
Glucuronic acid
Glucuronic acid-3,6-lactone
Glutamic acid
Glutamine
Glutaric acid
Glyceraldehyde-3-phosphate
Glyceric acid
Glyceric acid-2,3-diphosphate
Glyceric acid-3-phosphate
Glycerol
Glycerol-3-phosphate
Glycerol-beta-D-galactopyranoside
Glycine
Glycolic acid-2-phosphate
Glyoxylic acid
Gulonic acid
Gulonic acid-1,4-lactone
Gulose
Heptadecan-1-ol
Heptanoic acid
Hexadecanoic acid
Hexadecanol
Hexanoic acid
Homoserine
Hydroxyproline
Hydroxypropanoic acid
Hydroxytryptamine
Hypotaurine
Hypoxanthine
Idose
Iminodiacetic acid
Indole-2,3-dione
Indole-3-lactic acid
Inosine
Inositol
Isobutanoic acid
Isocitric acid
Isoleucine
Isomaltose
Itaconic acid
Ketoglutaric acid
Keto-L-gluconic acid
Lactic acid
Lactose
Leucine
Lysine
Lyxose
Maleamic acid
Maleic acid
Malic acid
Malonamide
Malonic acid
Maltose
Maltotriose
Table S2 (continued) List of putatively identified metabolites in MDA-MB-436, HMEC-1 and MCF-7 cell
lines across different applied sampling protocols
Mannitol
Mannose
Mannose-1-phosphate
Mannose-6-phosphate
Melatonin
Melezitose
Melibiose
Methionine
Methylmalonic acid
Methylthiopropylamine
myo-Inositol
myo-Inositol phosphate
N-Acetylglutamic acid
Nicotinamide
Nicotinic acid
Nonan-1-ol
Norleucine
Normetadrenaline
Norvaline
Octacosanol
Octadecadienoic acid
Octadecane
Octadecanoic acid
Octadecanol
Octadecenoic acid
Octan-1-ol
Octanoic acid
Ononitol
Ornithine
Ornithine-1,5-lactam
Orotic acid
Oxalic acid
Oxamic acid
Palatinose
Panthotenic acid
Pentadecan-1-ol
Pentadecane
Pentadecanoic acid
Pentanedioic acid
Pentanoic acid
Phenethylamine
Phenylalanine
Phosphomycin
Phosphoric acid
Phylloquinone
Picolinic acid
Pinitol
Pipecolic acid
Piperidinecarboxylic acid
Proline
Prolyl-glycine
Propane-1,2-diol
Purine
Putrescine
Pyroglutamic acid
Pyrrole-2-carboxylic acid
Pyruvic acid
Quinazoline
Raffinose
Resveratrol
Ribitol
Ribonic acid
Ribose
Sarcosine
Sedoheptulose
Sequoyitol
Serine
Sorbitol
Sorbitol-6-phosphate
Sorbose
Spermidine
Sphingosine
Suberic acid
Suberyl glycine
Succinic acid
Sucrose
Tagatose
Tetracosane
Tetradecane
Tetradecanoic acid
Threitol
Threonic acid
Threonic acid-1,4-lactone
Threonine
Thymine
Trehalose
Tridecan-1-ol
Triethanolamine
Tritriacontanol
Tryptamine
Tryptophan
Tyramine
Tyrosine
Undecanoic acid
Urea
Uric acid
Uridine
Valeric acid
Valine
Xylitol
Xylose