2 Instituto De Biologia Celular E Molecular (IBMC), University of Porto, 4150-180 Porto

Lipidomic approach to analyze phospholipid profiles and define class differences in mammary epithelial and breast cancer cells. M. Luísa Dória1, Zita Cotrim1, Bárbara Macedo2, Cláudia Simões1, Pedro Domingues1, Luísa Helguero1, and M. Rosário Domingues1

1 Mass Spectrometry Centre, Química Orgânica e Produtos Naturais (QOPNA), Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

2 Instituto de Biologia Celular e Molecular (IBMC), University of Porto, 4150-180 Porto, Portugal.

Supplementary Information

Supplementary table 1:Main features of mouse cell lines used

HD: hormone dependent

ND: not detected

*: Data extracted from Lanari C, et al, 2001(24)

**: Reichmann E, et al, 1992(23)

Supplementary Figure S1: Phosphatidylcholine (PC) MS/MS fragmentation

S1:ESI – MS/MS spectra of the [MH]+ at m/z 760 and [MNa]+ at m/z 782, corresponding to PC (16:00/18:01). Molecular structure of phosphatidylcholine (PC) is shown in the right presenting main cleavages corresponding to the fragmentation observed in the MS/MS spectrum of [MNa]+ ion. Fragmentation of [MH]+ ionsoriginated the characteristic ion of m/z 184 corresponding to the choline head of PCs (left, upper panel) and additional ions from [MNa]+providedmore structural information (left, lower panel)

Supplementary Figure S2:Sphingomyelin (SM) MS/MS fragmentation

S2:ESI – MS/MS spectra of the [MH]+ at m/z 703 and [MNa]+ at m/z 725, corresponding to SM (18:01/16:00). Molecular structure of Sphingomyelin (SM) is shown to the right presenting main cleavages corresponding to the fragmentation observed in the MS/MS spectrum of [MNa]+ ion. Fragmentation of [MH]+ ionsoriginated the characteristic ion of m/z 184 corresponding to the cholinehead of SMs (left, upper panel). Fragmentation of [MNa]+ ions gave more product ions thus providing additional structural information (left, lower panel), inclusive the ions corresponding to the loss of fatty acyl chain with water ([MNa-R2CO-H2O]+)

Supplementary Figure S3:Phosphatydilethanolamine (PE) MS/MS fragmentation

S3:ESI – MS/MS spectra of the [M+H]+ at m/z 746 and [M+Na]+ at m/z 768, corresponding to PE (18:00/18:01). Molecular structure of phosphatidylethanolamine (PE) is shown in the right presenting main cleavages correspondent to the fragmentation observed in the MS/MS spectrum of [M+Na]+ ion. Fragmentation of [M+H]+ forms a typical ion from the loss of 141 Da, characteristic of its ethanolamine head(left, upper panel). Fragmentation of [M+Na]+ ions gave more product ions thus providing additional structural information (left, lower panel).

Supplementary Figure S4:Phosphatydilserine (PS) MS/MS fragmentation

S4:ESI – MS/MS spectra of the [M-H]-at m/z 760, corresponding to PS (16:00/18:01). Molecular structure of phosphatidylserine (PS; top panel) presenting main cleavages corresponding to the fragmentation observed in the MS/MS spectrum of [M-H]- ion. Fragmentation of [M-H]- gave the typical loss of m/z 87 ([M-H-87]-), characteristic of its serine polar head, and ions corresponding to the fatty acyls chains ([R2COO]- and [R1COO]-; bottom panel).

Supplementary Figure S5:Phosphatydilinositol (PI) MS/MS fragmentation

S5:ESI – MS/MS spectra of the [M-H]-at m/z 837, corresponding to PI (16:00/18:00). Molecular structure of phosphatidylinositol (PI; top panel) presenting main cleavages corresponding to the fragmentation observed in the MS/MS spectrum of [M-H]- ion. Fragmentation of [M-H]- gave the typical ions as the loss of fatty acyls with the inositol ([M-H-R1COOH-162]- and [M-H-R2COOH-162]-), loss of fatty acyls chains ([M-H-R1COOH]- and [M-H-R2COOH]-), and ions corresponding to the fatty acyls chains ([R2COO]- > [R1COO]-; bottom panel)

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