1. Supplementary Material
Journal of Proteomics
Supplementary Material
Stable isotope labeling by fatty acids in cell culture (SILFAC) coupled with isotope pattern dependent mass spectrometry for global screening of lipid hydroperoxide-mediated protein modifications
Ryo Takahashi, Shuhei Fujioka, Tomoyuki Oe* and Seon Hwa Lee*
Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences,
Tohoku University, Sendai, Miyagi , Japan
*Corresponding authors: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai , Japan.
Phone: Fax:

2. Table S1. 13-HPODE-derived modifications to HSA identified by LC/ESI-DDS/MS analysis. * indicates a modification site.
a detected more than two times in three separate experiments with <1% FDR.

3. Table S2. Proteins in MCF-7 cells (control) identified by LC/ESI-DDS/MS.
Coverage
Triosephosphate isomerase
91.2
78 kDa glucose-regulated protein
61.6
Peptidyl-prolyl cis-trans isomerase A
85.5
ADP-ribosylation factor 3
61.3
Fructose-bisphosphate aldolase A
82.7
Tubulin-specific chaperone A
61.1
Cellular retinoic acid-binding protein 2
82.6
40S ribosomal protein S13
60.9
Tubulin beta chain
82.2
Tubulin beta-3 chain
60.7
Tubulin beta-2C chain
82.0
40S ribosomal protein S23
60.1
Phosphoglycerate kinase 1
79.4
protein theta
60.0
Cofilin-1
78.3
Transgelin-2
59.8
Alpha-enolase
77.4
Tropomyosin alpha-4 chain
59.7
60S acidic ribosomal protein P2
Heat shock 70 kDa protein 1A/1B
59.4
protein epsilon
76.1
40S ribosomal protein S15a
59.2
Heat shock protein beta-1
75.6
Activated RNA polymerase II transcriptional coactivator p15
59.1
Peroxiredoxin-1
75.4
Chloride intracellular channel protein 1
58.9
Pyruvate kinase isozymes M1/M2
75.3
40S ribosomal protein S19
58.6
40S ribosomal protein S3
74.9
40S ribosomal protein S21
57.8
Actin, cytoplasmic 1
74.4
Tubulin alpha-1C chain
57.7
60 kDa heat shock protein, mitochondrial
74.0
40S ribosomal protein S12
57.6
10 kDa heat shock protein, mitochondrial
73.5
Tubulin alpha-1A chain
57.4
protein zeta/delta
Hypoxanthine-guanine phosphoribosyltransferase
57.3
Peroxiredoxin-6
73.2
Brain acid soluble protein 1
Poly(rC)-binding protein 1
71.9
Na(+)/H(+) exchange regulatory cofactor NHE-RF1
Phosphatidylethanolamine-binding protein 1
71.7
Protein disulfide-isomerase A3
57.2
Protein DJ-1
71.4
Tubulin alpha-1B chain
Profilin-1
70.7
60S acidic ribosomal protein P1
57.0
Ras-related protein Rab-1B
70.2
Keratin, type I cytoskeletal 18
56.5
Glucose-6-phosphate 1-dehydrogenase
70.1
Heterogeneous nuclear ribonucleoprotein K
56.4
BolA-like protein 2
69.8
Heat shock protein HSP 90-beta
Keratin, type II cytoskeletal 8
69.6
Myosin light polypeptide 6
56.3
60S ribosomal protein L30
40S ribosomal protein S4, X isoform
Superoxide dismutase
69.5
ATP synthase subunit beta, mitochondrial
56.1
Nucleophosmin
67.7
Calreticulin
55.6
Malate dehydrogenase, mitochondrial
67.5
Annexin A5
Translationally-controlled tumor protein
67.4
Heat shock protein HSP 90-alpha
55.3
Nucleoside diphosphate kinase B
67.1
Electron transfer flavoprotein subunit alpha, mitochondrial
3-hydroxyacyl-CoA dehydrogenase type-2
Ribonuclease inhibitor
55.1
Phosphoglycerate mutase 1
66.9
Heat shock cognate 71 kDa protein
54.6
Galectin-1
65.9
Peroxiredoxin-2
Annexin A2
65.8
Thymosin beta-4-like protein 3
Thioredoxin
65.7
Fructose-1,6-bisphosphatase 1
54.4
Keratin, type I cytoskeletal 19
65.0
Proteasome activator complex subunit 1
54.2
Nucleoside diphosphate kinase A
64.5
Elongation factor 2
53.9
Ras-related protein Rab-1A
64.4
Transcription intermediary factor 1-beta
53.8
40S ribosomal protein S3a
64.0
Hematological and neurological expressed 1-like protein
53.7
Protein disulfide-isomerase
protein gamma
53.4
Peptidyl-prolyl cis-trans isomerase B
63.0
60S ribosomal protein L37a
53.3
40S ribosomal protein S17
protein beta/alpha
Glyceraldehyde-3-phosphate dehydrogenase
62.7
Rab GDP dissociation inhibitor beta
53.0
40S ribosomal protein S11
14 kDa phosphohistidine phosphatase
52.8
40S ribosomal protein S8
62.5
60S ribosomal protein L12
52.7

4. Table S3. Proteins in MCF-7 cells (treated with A23187 and AscA) identified by LC/ESI-DDS/MS.
Coverage
Triosephosphate isomerase
88.4
Malate dehydrogenase, mitochondrial
60.1
Cellular retinoic acid-binding protein 2
82.6
BolA-like protein 2
59.3
protein zeta/delta
81.2
Nucleophosmin
57.8
60S acidic ribosomal protein P2
80.0
40S ribosomal protein S8
57.7
Tubulin beta chain
79.3
Thioredoxin
57.1
Alpha-enolase
Heterogeneous nuclear ribonucleoprotein K
56.8
Tubulin beta-2C chain
79.1
Tubulin alpha-1C chain
Keratin, type II cytoskeletal 8
76.8
Transcription intermediary factor 1-beta
Peroxiredoxin-1
76.4
Tubulin alpha-1B chain
Cofilin-1
75.9
3-hydroxyacyl-CoA dehydrogenase type-2
56.3
60 kDa heat shock protein, mitochondrial
75.6
Peroxiredoxin-5, mitochondrial
56.1
Pyruvate kinase isozymes M1/M2
75.3
ATP synthase subunit beta, mitochondrial
56.0
Actin, cytoplasmic 1
74.4
40S ribosomal protein S23
55.9
Phosphoglycerate kinase 1
73.1
Superoxide dismutase [Cu-Zn]
55.8
Fructose-bisphosphate aldolase A
Proteasome activator complex subunit 1
55.4
40S ribosomal protein S3
72.8
Chloride intracellular channel protein 1
54.8
Nucleoside diphosphate kinase B
71.7
Heat shock protein HSP 90-alpha
54.6
10 kDa heat shock protein, mitochondrial
71.6
40S ribosomal protein S4, X isoform
54.4
Profilin-1
70.7
Heat shock 70 kDa protein 1A/1B
53.8
Peroxiredoxin-6
70.1
40S ribosomal protein S19
Peptidyl-prolyl cis-trans isomerase B
69.9
Protein disulfide-isomerase A3
53.3
60S ribosomal protein L30
69.6
60S ribosomal protein L37a
Tubulin-specific chaperone A
69.4
Elongation factor 2
53.2
protein epsilon
69.0
Myosin light polypeptide 6
53.0
Glucose-6-phosphate 1-dehydrogenase
67.8
40S ribosomal protein S13
Keratin, type I cytoskeletal 19
67.5
60S ribosomal protein L7
52.8
60S ribosomal protein L12
67.3
Ras-related protein Rab-1B
52.7
ADP-ribosylation factor 3
66.9
60S ribosomal protein L35a
51.8
Keratin, type I cytoskeletal 18
66.3
Fructose-1,6-bisphosphatase 1
51.5
Proliferating cell nuclear antigen
65.9
Transitional endoplasmic reticulum ATPase
51.4
Protein disulfide-isomerase
65.4
Electron transfer flavoprotein subunit alpha, mitochondrial
Glyceraldehyde-3-phosphate dehydrogenase
65.1
Tumor protein D54
51.0
Peptidyl-prolyl cis-trans isomerase A
64.9
Glutathione S-transferase Mu 3
50.7
40S ribosomal protein S11
64.6
Hematological and neurological expressed 1-like protein
50.5
Heat shock protein beta-1
64.4
Na(+)/H(+) exchange regulatory cofactor NHE-RF1
50.3
Transgelin-2
64.3
Ras-related protein Rab-1A
49.8
Annexin A2
64.0
40S ribosomal protein S16
49.3
protein beta/alpha
63.4
40S ribosomal protein S15a
49.2
40S ribosomal protein S3a
63.3
Endoplasmin
48.4
Poly(rC)-binding protein 1
63.2
Small nuclear ribonucleoprotein Sm D2
48.3
Phosphatidylethanolamine-binding protein 1
63.1
T-complex protein 1 subunit beta
48.2
Tropomyosin alpha-4 chain
62.9
Calreticulin
48.0
protein theta
Activated RNA polymerase II transcriptional coactivator p15
47.2
Translationally-controlled tumor protein
62.8
Tubulin beta-3 chain
47.1
Protein DJ-1
62.4
Elongation factor 1-delta
47.0
60S acidic ribosomal protein P1
62.3
Calmodulin
78 kDa glucose-regulated protein
61.0
Rab GDP dissociation inhibitor beta
Heat shock protein HSP 90-beta
60.8
Stress-induced-phosphoprotein 1
Phosphoglycerate mutase 1
60.2
Chromobox protein homolog 3
46.5

5. A B
Fig. S1. Structures of (A) PC and (B) PE (R1 = palmitic acid (16:0) or stearic acid (18:0), R2 = LA or [13C18]-LA).

6. 283.3 (sn-1)
279.2 (sn-2)
770.6
255.2 (sn-1)
742.6 A B C D
Fig. S2. Fragmentation patterns of (A) PLPC, (B) PLPE, (C) SLPC, and (D) SLPE in the negative ion mode.

7. relative intensity (%)
714.4
[M−H]−
782.2
279.0
[C18H31O2]−
255.0
[C16H31O2]−
m/z
802.2
[M+HCO2]−
742.2
[M−CH3]−
279.2
255.2 A B C D 50
100
200
300
400
500
600
700
800
900
1000
5.4E6
9.0E6
CE 40
3.4E6
6.0E6
CE 45
Fig. S3. MS and MS/MS analyses of PLPC and PLPE in the negative ion mode. (A) Full scan spectrum of PLPC, (B) MS2 spectrum of m/z (PLPC), (C) Full scan spectrum of PLPE, and (D) MS2 spectrum of m/z (PLPE).

8. relative intensity (%)50
100
742.2
[M−H]−
810.2
279.1
[C18H31O2]−
2.5E6
5.2E6
CE 40
m/z
200
300
400
500
600
700
800
900
1000
830.3
[M+HCO2]−
770.3
[M−CH3]−
279.0
3.3E6
5.9E6
CE 45
relative intensity (%)
283.2
[C18H35O2]−
283.3 A B C D
Fig. S4. MS and MS/MS analyses of SLPC and SLPE in the negative ion mode. (A) Full scan spectrum of SLPC, (B) MS2 spectrum of m/z (SLPC), (C) Full scan spectrum of SLPE, and (D) MS2 spectrum of m/z (SLPE).

9. 16.3
16.2
16.1
2.0E6
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
0 h
6 h
24 h
72 h
120 h
240 h
Fig. S5. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for PLPC were m/z → (12C18) and m/z → (13C18).

10. 16.4
16.3
16.2
2.0E5
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
0 h
6 h
24 h
72 h
120 h
240 h
Fig. S6. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for PLPE were m/z → (12C18) and m/z → (13C18).

11. 16.3
16.4
16.2
5.0E5
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
0 h
6 h
24 h
72 h
120 h
240 h
Fig. S7. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for PLPE were m/z → (12C18) and m/z → (13C18).

12. 21.6
21.7
21.5
21.4
1.0E5
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
0 h
6 h
24 h
72 h
120 h
240 h
Fig. S8. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for SLPC were m/z → (12C18) and m/z → (13C18).

13. 21.6
21.7
21.5
21.4
3.0E5
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
0 h
6 h
24 h
72 h
120 h
240 h
Fig. S9. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for SLPC were m/z → (12C18) and m/z → (13C18).

14. 21.7
21.6
21.5
3.0E5
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
0 h
6 h
24 h
72 h
120 h
240 h
Fig. S10. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for SLPE were m/z → (12C18) and m/z → (13C18).

15. 21.7
21.6
retention time (min) 10 20 50
100
12C18
13C18
relative intensity (%)
21.5
0 h
6 h
24 h
72 h
120 h
240 h
1.0E6
Fig. S11. Time course analysis of [13C18]-LA incorporation. Samples were obtained at 0, 6, 24, 72, 120, and 240 h. SRM transitions for SLPE were m/z → (12C18) and m/z → (13C18).

16. Abs (570 nm)
control
A23187+AscA
Fig. S12. Cell viability evaluated by MTT assay. MCF-7 cells were incubated with or without 1 μM A23187 and 1 mM AscA in DMEM (FBS free) at 37 oC for 24 h. Results are presented as means ± SEM for five independent experiments.