46BR.1N fibroblasts Proteomic Analysis Results

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Preliminary analysis of the proteomic dataset

The aim of this study was to investigate the proteins related to the AA cascade using SWATH-MS. As this was a preliminary study, the results are presented as raw intensities.

To investigate the effect of treatment on protein expression, cells stimulated with A23187 (5 µM, 6h), UVR (15 mJ/cm2, 24h) and unstimulated cells were analysed. Peptides were detected for all proteins responsible for the production of lipid mediators detected by LC-MS/MS. No peptides were detected for the UVR inducible proteins (COX-2 and mPGES-1) in the irradiated samples. The protein that was detected at the highest mean intensity and the most consistently was PTGR-2, the prostaglandin reductase protein. The most inconsistent protein was PGFS (AKR13), the aldo-keto reductase family 1-member C3 isoform of prostaglandin F synthase. When the different treatments were compared, no difference could be found.

Proteomic analysis of AA cascade proteins in 46BR.1N fibroblasts. Results shown as the raw intensities from three independent experiments of unstimulated (red circle), A23187 stimulated (green triangle) and UVR stimulated 46BR.1N fibroblasts (blue diamond).

The raw intensity of proteins detected in 46BR.1N fibroblasts which were unstimulated and stimulated with A23187 and UVR. Data is presented as mean ± SD for dual injections of three independent experiments.

	Unstimulated			A23187 (5 mM, 6h)			UVR (15 mJ/cm2, 6h)
	Mean	SD	n	Mean	SD	n	Mean	SD	n
12(S)-LOX	3.89E+03	1.92E+03	4	5.77E+03	0.00E+00	1	5.65E+03	1.46E+03	3
15-LOX-1	8.52E+03	5.21E+03	4	9.18E+03	5.18E+03	4	9.44E+03	1.15E+03	4
15-PGDH	1.16E+03	6.06E+02	2	1.75E+03	1.40E+03	4	1.79E+03	3.80E+02	2
5-LOX	1.17E+03	0.00E+00	1	5.30E+01	0.00E+00	1	1.36E+03	1.17E+03	4
5LOX/FLAP	9.06E+02	0.00E+00	1	1.93E+03	1.18E+03	3	8.32E+02	6.92E+02	2
COX-1	2.36E+03	2.40E+03	4	4.66E+03	2.58E+03	3	6.36E+03	2.86E+03	4
cPGES	2.13E+05	8.09E+04	5	2.31E+05	1.42E+05	6	2.30E+05	9.48E+04	6
HPGDS	6.01E+03	0.00E+00	1	6.67E+03	5.27E+03	3	4.04E+03	2.20E+03	4
LTA4H	1.64E+04	5.48E+03	5	1.42E+04	3.14E+03	6	1.64E+04	2.95E+03	6
mPGES-2	1.22E+04	9.04E+03	5	8.36E+03	6.24E+03	6	4.52E+03	2.34E+03	6
PGFS (AKR13)	2.34E+03	1.39E+03	3	1.90E+03	0.00E+00	1	1.32E+03	0.00E+00	1
PHGPx	2.90E+03	2.18E+03	4	2.17E+03	1.55E+03	4	1.47E+03	4.73E+02	3
PTGR2	2.29E+04	3.68E+04	6	4.49E+05	5.19E+05	5	2.94E+05	5.95E+05	6
TXAS	2.89E+03	2.41E+03	3	1.70E+03	1.38E+03	3	1.94E+03	2.02E+03	2

Further analysis of the proteomic dataset

This analysis, performed by the Stoller centre, included taking the top three most intense peptide species, a 1% false discovery rate and acceptance of transition groups if it was identified in the majority of replicates (Table *, Figure *). This in-depth analysis only found five proteins related to the eicosanoid cascade. PHGPx and LTA4H were consistently identified in 7/9 samples. The isoform of PGES which was detected was not specified.

In-depth proteomic analysis of AA cascade proteins in 46BR.1N fibroblasts. Results shown are from three independent experiments of unstimulated (red circle), A23187 stimulated (green triangle) and UVR stimulated 46BR.1N fibroblasts (blue diamond).

The Log2 intensity of proteins detected in 46BR.1N fibroblasts which were unstimulated and stimulated with A23187 and UVR. Data is presented as mean ± SD for three independent experiments.

Proteins	Unstimulated			A23187 (5 µM, 6h)			UVR (15 mJ/cm2, 24h)
	Mean	SD	n	Mean	SD	n	Mean	SD	n
COX-1	11.94	0	1				10.8	0	1
LTA4H	10.01	0.29	2	9.81	0.92	3	5.39	7.62	2
PGES	9.6	0.48	3	9.7	0.29	2	9.85	0	1
PHGPx	10.38	0.32	3	8.58	1.34	3	9.59	0	1
TXAS	8.01	1.4	2	3.06	5.3	3