International Journal of Proteomics

Research Article

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

Table 1

Changes in abundance and S-nitrosylation proteome profile of PBMC proteins in human heart failure.


2D-GE data			MALDI MS data				²ASC⁻/ASC⁺ (ΔSNO) ratio		¹Abundance (ASC⁺)	³S-NO ROR
Spot number	pI	MW (kD)	Plate number	Protein and gene name	Accession number	MS (LC MS/MS) ID protein score	NH	HF	HF versus NH	HF versus NH

52	6.39	108	F1	Vinculin GN=VCL	P18206	709	1.14	−1.03	−1.88	1.83
54	6.57	108	H14	Vinculin GN=VCL	P18206-2	1180	1.08	1.09	−3.29	3.58
57	6.68	107	H15	Vinculin GN=VCL	P18206-2	1130	−1.03	−1.06	−3.09	2.91
58	6.80	107	F20	Vinculin GN=VCL	P18206-2	1210	−1.01	1.04	−3.61	3.76
59	6.94	107	F21	Vinculin GN=VCL	P18206	1170	1.01	1.04	−3.27	3.39
60	7.09	105	C14	FAM212B GN=FAM212B	Q9NTI7	29	1.04	−1.06	−2.38	2.25
63	7.53	99	7	Vinculin GN=VCL	P18206	236	1.12	−1.24	−1.54	1.24
79	6.48	96	F22	Albumin GN=ALB	P02768	190	−1.28	−1.14	−2.03	1.78
81	6.62	94	F23	Keratin, type I cytoskeletal 9 GN=KRT9	P35527	188	−1.18	−1.23	−2.37	1.93
92	6.93	87	F24	Filamin-A GN=FLNA	Q5HY54	384	−1.17	−1.56	−1.20	−1.31
95	6.81	87	G2	Filamin-A GN=FLNA	Q5HY54	399	−1.19	−1.22	−2.02	1.66
99	4.54	84	G4	Glucose-regulated 78 kDa protein GN=HSPA5	P11021	849	1.01	1.29	−1.61	2.08
102	6.68	83	G5	Filamin-A GN=FLNA	Q5HY54	210	−1.26	−1.32	−1.64	1.24
103	6.41	82	H21	Filamin-A GN=FLNA	P21333	238	−1.28	−1.03	−1.68	1.63
104	6.52	82	I20	Gelsolin GN=GSN	P06396-2	182	−1.32	−1.11	−1.72	1.55
124	5.21	68	G6	Heat shock 71 kDa protein GN=HSPA8	P11142-2	325	1.04	−1.01	−1.52	1.51
127	9.09	68	G7	Fibrinogen alpha chain GN=FGA	P02671-2	62	1.07	−1.65	1.54	−2.53
131	5.35	68	G8	Keratin, type I cytoskeletal 10 GN=KRT10	P13645	296	−1.15	−1.08	−1.72	1.59
136	8.84	68	E6	Transketolase GN=TKT	B4E022	189	−1.03	−1.88	1.84	−3.46
141	5.97	63	1	Serum albumin GN=ALB	H0YA55	167	−1.14	1.09	1.07	1.03
142	4.51	63	G9	Tropomyosin 3 GN=TPM3	Q5VU59	743	1.01	1.34	−1.83	2.46
145	6.41	60	A1	Serum albumin GN=ALB	H0YA55	245	−1.59	−1.28	−1.04	−1.24
146	6.78	60	C15	Serum albumin GN=ALB	P02768	116	−1.00	−1.42	1.32	−1.88
154	5.34	59	H24	Actin, cytoplasmic 1 GN=ACTB	P60709	391	−1.43	−1.07	−1.55	1.44
180	5.14	54	J11	Actin, cytoplasmic 1 GN=ACTB	P60709	353	−1.14	1.18	−1.76	2.07
185	5.50	53	K19	Actin, cytoplasmic 1 GN=ACTB	P60709	365	1.28	−1.16	1.41	−1.63
197	7.48	51	G12	Fibrinogen beta chain GN=FGB	P02675	157	−1.14	−1.07	−1.53	1.43
204	4.29	50	G13	Nucleosome assembly protein 1 GN=NAP1L1	F8VRJ2	79	1.21	−1.07	−1.74	1.62
205	8.50	50	C16	Fibrinogen beta chain GN=FGB	P02675	336	−1.11	−1.18	1.00	−1.18
222	4.71	47	J13	ATP synthase subunit beta GN=ATP5B	H0YH81	302	−1.06	1.20	−1.92	2.30
241	8.79	45	J1	Transcription factor 4 GN=TCF4 (integrin-linked protein kinase GN=ILK)	H3BME8	36 (305)	−1.73	−1.16	−1.68	1.45
261	6.57	41	101	POTE ankyrin domain family member F	A5A3E0	121	−1.47	1.16	−1.35	1.57
262	7.68	41	I5	Elongation factor Tu, mitochondrial GN=TUFM	P49411	195	−1.52	−1.04	−1.58	1.52
264	4.86	41	J18	Actin, cytoplasmic 1 GN=ACTB	P60709	336	−1.05	1.06	−1.49	1.58
267	6.41	41	16	Actin, cytoplasmic 2, N-terminal	F5H0N0	106	−1.19	1.33	−1.15	1.53
272	8.70	41	B9	Myeloperoxidase GN=MPO	P05164-2	83	−1.30	−1.47	1.22	−1.79
303	4.54	38	B10	Actin, cytoplasmic 1 GN=ACTB	P60709	91	1.17	1.18	−1.32	1.55
329	7.17	35	I10	DnaJ homolog subfamily B 11 GN=DNAJB11	H7C2Y5	41	−1.25	−1.46	1.17	−1.70
335	5.55	35	D6	Actin, cytoplasmic 1 GN=ACTB	P60709	225	−1.14	−1.09	1.88	−2.04
337	5.81	34	D7	Actin, cytoplasmic 1 GN=ACTB	P60709	456	−1.20	−1.06	1.64	−1.74
339	6.33	34	105	Tubulin beta chain GN=TUBB	Q5JP53	121	−1.28	−1.38	1.19	−1.64
343	6.72	34	B13	Actin, cytoplasmic 1 GN=ACTB	P60709	97	−1.17	−1.36	1.26	−1.71
344	3.63	33	A2	Stromal interaction molecule 2 GN=STIM2	Q9P246	33	1.43	−1.18	1.98	−2.34
348	7.11	33	D8	Phosphoglycerate kinase GN=PGK1	B7Z7A9	54	−1.23	−1.92	1.49	−2.86
358	4.56	31	G24	Tropomyosin 1 alpha isoform 7 GN=TPM1	D9YZV8	515	1.20	1.13	−1.52	1.72
373	7.32	30	H1	Annexin A1 GN=ANXA1	P04083	200	−1.18	−2.15	−1.13	−1.91
384	4.02	29	L10	LVV-hemorphin-7 GN=HBB	F8W6P5	47	−1.09	1.57	−1.98	3.12
385	5.72	29	102	Vimentin GN=VIM	F5H288	171	−1.22	−1.36	1.20	−1.63
395	6.85	27	E8	Keratin, type II cytoskeletal 2 GN=KRT2	P35908	381	−1.46	−1.65	1.70	−2.80
397	4.01	27	J23	Tropomyosin alpha-4 chain GN=TPM4	P67936	101	1.07	1.41	−1.58	2.22
400	6.70	26	L2	Thrombospondin-1 GN=THBS1	P07996	769	−1.14	1.05	2.00	−1.91
404	7.51	26	17	Actin, cytoplasmic 1, N-terminal GN=ACTB	B4DW52	133	1.23	−1.09	1.91	−2.08
406	8.08	26	K1	Uncharacterized protein FLJ46347	Q6ZRH9	36	−1.07	−1.16	1.88	−2.18
411	7.98	26	5	Myosin-IXa GN=MYO9A	H3BMM1	46	1.09	−1.27	2.26	−2.86
424	4.69	25	F15	Tropomyosin alpha-4 chain GN=TPM4	P67936	926	1.73	1.18	1.85	−1.57
425	8.63	25	12	Peptidyl-prolyl cis-trans isomerase A GN=PPIA	P62937	110	−1.36	−1.17	1.46	−1.71
431	6.70	24	H3	Haloacid dehalogenase-like hydrolase domain-containing protein 2 GN=HDHD2	K7ER15	102	−1.20	−1.27	1.42	−1.80
451	9.33	23	F16	Glyceraldehyde-3-phosphate dehydrogenase GN=GAPDH	E7EUT4	71	1.02	−1.36	1.12	−1.52
461	6.98	22	B17	Annexin A1 GN=ANXA1	P04083	344	1.23	−1.07	1.55	−1.65
476	4.74	21	D10	Actin, cytoplasmic 1 GN=ACTB	P60709	226	1.05	1.22	1.51	−1.24
491	8.58	20	B18	Thrombospondin-1 GN=THBS1	P07996	138	1.20	−1.20	2.45	−2.95
501	6.78	20	H6	Peroxiredoxin-6 GN=PRDX6	P30041	237	−1.08	−1.48	1.51	−2.23
505	8.22	20	I14	Thrombospondin-1 GN=THBS1	P07996	197	1.06	1.07	1.52	−1.42
507	6.61	20	H7	Growth factor receptor-bound protein 2 GN=GRB2	P62993-2	80	−1.01	−1.14	1.48	−1.68
509	8.72	20	L3	Thrombospondin-1 GN=THBS1	P07996	418	−1.03	−1.29	2.74	−3.53
511	7.30	20	D14	Carnitine O-palmitoyltransferase 1 GN=CPT1A	P50416	32	−1.15	−1.03	1.65	−1.70
514	4.10	20	E9	Transcriptional repressor protein YY1 GN=YY1	H0YJV7	47	1.01	1.27	−1.43	1.82
515	5.26	19	L4	Apolipoprotein A-I GN=APOA1	P02647	352	1.06	1.11	1.56	−1.40
518	4.29	19	C21	Vimentin GN=VIM	B0YJC4	203	1.13	1.37	−1.17	1.60
524	9.34	19	108	Keratin, type I cytoskeletal 10 GN=KRT10	P13645	286	−1.02	−1.47	1.42	−2.08
530	8.61	19	L11	Keratin, type II cytoskeletal 1 GN=KRT1	P04264	55	1.42	−1.35	3.00	−4.04
549	6.94	19	J5	Glutathione GN=GSH (Ras-related protein Rab-14 GN=RAB14)	P07203	71 (288)	1.10	−1.13	1.57	−1.78
563	5.99	18	109	Actin, cytoplasmic 2, N-terminal GN=ACTB	I3L1U9	137	−1.53	1.07	−1.60	1.71
567	6.93	18	C22	Glutathione S-transferase P GN=GSTP1	P09211	286	−1.02	−1.30	1.26	−1.64
568	3.62	18	E10	Transcriptional repressor protein YY1 GN=YY1	H0YJV7	39	1.47	−1.22	1.83	−2.24
574	4.65	17	C23	Tubulin beta-1 chain GN=TUBB1	Q9H4B7	39	1.21	1.28	−2.09	2.66
588	3.88	17	A8	Alpha-actinin-1 GN=ACTN1 (Alpha actinin 1 GN=ACTN1)	H0YJ11	50 (222)	1.44	1.24	−1.28	1.59
591	7.55	17	E12	Heat shock 70 kDa protein 1A/1B GN=HSPA1A	E7EP11	43	1.01	−1.07	1.43	−1.52
592	4.38	17	106	Myosin regulatory light chain 12B GN=MYL12B	O14950	320	1.43	1.62	−1.71	2.78
593	4.47	17	C24	Actin, cytoplasmic 1 GN=ACTB	P60709	65	1.63	1.75	−1.66	2.91
595	8.27	17	A9	Protein S100-A8 GN=S100A8	P05109	60	1.59	−1.07	1.65	−1.76
603	4.76	16	A10	Annexin A5 GN=ANXA5	D6RBL5	111	−1.20	−1.37	1.13	−1.55
605	5.64	16	110	ATP synthase subunit alpha GN=ATP5A1	A8K092	95	−1.03	−1.00	−1.02	1.01
607	6.71	16	B20	Actin-related protein 2/3 complex subunit 3 GN=ARPC3	O15145	50	1.06	1.14	1.64	−1.43
612	4.19	16	K21	Myosin regulatory light chain 9 GN=MYL9	P24844	233	1.19	1.38	1.62	−1.17
613	4.61	16	D17	Actin, cytoplasmic 2 GN=ACTG1	P63261	61	1.45	1.21	1.78	−1.47
623	5.16	16	A12	Actin, cytoplasmic 2, N-terminal GN=ACTG1	K7EM38	130	1.30	1.29	1.87	−1.45
627	5.47	15	3	Annexin A6 GN=ANXA6	E5RIU8	103	−1.25	1.03	1.30	−1.26
628	3.79	15	E14	Myosin light polypeptide 6 GN=MYL6	F8VZV5	302	1.30	1.32	1.12	1.18
629	4.35	15	A13	Actin, cytoplasmic 1 GN=ACTB	P60709	65	1.24	1.32	1.15	1.14
630	8.05	15	K6	Peptidyl-prolyl cis-trans isomerase A GN=PPIA	P62937	199	−1.04	−1.18	−1.10	−1.07
632	9.02	15	I15	Peptidyl-prolyl cis-trans isomerase A GN=PPIA	P62937	235	−1.01	−1.12	−1.20	1.07
640	5.12	15	13	Actin, cytoplasmic 1, N-terminal GN=ACTB	G5E9R0	193	1.45	1.40	1.82	−1.30
642	4.21	15	E15	Actin, cytoplasmic 1 GN=ACTB	P60709	139	1.05	1.25	−1.31	1.64
644	7.71	15	21	Keratin, type I cytoskeletal 10 GN=KRT10	P13645	47	1.16	1.32	−1.07	1.41
646	3.98	15	H9	Myosin light polypeptide 6 GN=MYL6	F8VZV5	369	1.16	1.41	−1.39	1.97
648	8.37	15	D1	Bestrophin-3 GN=BEST3 (dermcidin GN=DCD)	A8MVM3	42 (160)	1.17	1.98	1.29	1.53
657	4.79	15	A14	Actin, cytoplasmic 1 GN=ACTB	P60709	108	1.25	1.12	1.62	−1.44
662	4.65	14	A16	Actin, cytoplasmic 1 GN=ACTB	P60709	263	1.55	1.62	1.65	−1.02
664	7.01	14	D19	Actin, cytoplasmic 2, N-terminal GN=ACTG1	K7EM38	41	1.53	1.09	1.29	−1.18
665	4.45	14	A17	Actin, cytoplasmic 1 GN=ACTB	P60709	233	1.56	1.16	1.51	−1.30
666	3.82	14	B22	Mitochondrial carrier homolog 1 GN=MTCH1	Q9NZJ7-3	32	1.34	1.40	−1.01	1.42
671	8.66	14	B23	Bestrophin-3 GN=BEST3	A8MVM3	40	1.21	1.43	2.00	−1.40
673	4.03	14	B24	Isoform H14 of Myeloperoxidase GN=MPO	P05164-2	288	−1.16	1.18	−1.48	1.76
683	5.01	13	A22	Histone H4 GN=HIST1H4A	P62805	242	1.01	−1.09	1.44	−1.56
690	7.92	13	L13	Hemoglobin subunit beta GN=HBB	P68871	384	−1.15	−1.12	1.50	−1.68
703	7.72	12	D3	LVV-hemorphin-7 (fragment) GN=HBB	F8W6P5	41	−1.13	1.09	−1.49	1.63
706	6.70	12	E16	Protein S100-A11 GN=S100A11	P31949	260	−1.39	1.27	−1.75	2.22
718	4.53	11	C4	Ras-related protein Ral-B GN=RALB (actin cytoplasmic 2 GN=ACTG1)	F8WEQ6	48 (155)	1.20	−1.30	1.64	−2.14
732	4.72	11	C6	Thrombospondin-1 GN=THBS1	P07996	115	1.61	−2.02	2.50	−5.05
737	4.00	10	D20	ATP synthase subunit alpha GN=ATP5A1	K7EQT2	173	−1.16	−1.90	2.40	−4.57
738	3.88	10	D21	Keratin, type II cytoskeletal 1 GN=KRT1	P04264	96	1.07	−1.46	1.85	−2.70
740	4.86	0	C7	Urea transporter 1 GN=SLC14A1	K7EJ54	39	1.33	−1.35	1.78	−2.40
744	4.40	0	23	Ras-related protein 1b GN=RAP1B	B4DQI8	58	−1.02	−1.30	1.81	−2.35
759	4.82	15	D22	Actin, cytoplasmic 2, N-terminal GN=ACTG1	K7EM38	263	1.27	1.37	1.73	−1.27
761	4.82	16	D23	Keratin, type II cytoskeletal 1 GN=KRT1	P04264	789	1.24	1.31	1.62	−1.24
762	4.91	16	L14	Actin, cytoplasmic 2 GN=ACTG1	P63261	221	1.34	1.18	1.69	−1.44
763	5.73	20	A24	Actin, cytoplasmic 1 GN=ACTB	P60709	416	−1.06	−1.09	1.89	−2.05
769	5.38	14	B1	Actin, cytoplasmic 1 GN=ACTB	C9JUM1	44	1.07	−1.05	1.59	−1.67
772	5.37	13	B2	Histone H4 GN=HIST1H4A	P62805	193	−1.36	−1.36	1.13	−1.54
774	3.72	16	J7	Calmodulin GN=CALM1	P62158	367	−1.11	1.47	−1.51	2.23
779	4.63	28	D24	Actin, cytoplasmic 1 GN=ACTB	P60709	428	1.16	1.21	−1.28	1.55
781	4.61	27	E1	Actin, cytoplasmic 1 GN=ACTB	P60709	342	1.04	1.01	−1.07	1.08
796	5.29	13	E19	Keratin, type II cytoskeletal 1 GN=KRT1	P04264	127	1.29	−1.70	2.35	−4.00
797	5.34	13	E20	Keratin, type II cuticular Hb3 GN=KRT83	P78385	165	1.08	−1.11	1.59	−1.77
804	6.96	10	E21	LVV-hemorphin-7 (fragment) GN=HBB	F8W6P5	122	1.26	1.36	−1.40	1.90
808	8.11	11	L6	Platelet basic protein GN=PPBP	P02775	90	1.43	1.19	−1.38	1.64
825	8.65	72	C8	Lactotransferrin delta GN=LTF	P02788-2	147	−1.44	−1.65	1.51	−2.49
854	8.74	67	C9	Kaliocin-1 GN=LTF	E7EQB2	46	−1.09	−1.50	1.22	−1.83
866	4.88	10	C10	S100-A6 protein GN=S100A6	P06703	39	1.46	−1.12	1.94	−2.18
867	4.91	0	C11	LVV-hemorphin-7 GN=HBB	F8W6P5	65	1.26	1.16	1.50	−1.29
868	3.59	16	I18	Calmodulin GN=CALM1	P62158	343	−1.01	1.27	−1.70	2.16
870	3.64	16	K10	Calmodulin GN=CALM1 (calmodulin GN=CALM1)	E7ETZ0	63 (215)	1.45	1.60	−1.34	2.15
871	3.69	16	K11	Calmodulin GN=CALM1	E7ETZ0	195	1.33	1.31	−1.49	1.95
877	3.80	55	B4	Stromal interaction molecule 2 GN=STIM2	Q9P246	39	1.31	−1.30	1.69	−2.20
879	6.87	11	E23	LVV-hemorphin-7 GN=HBB	F8W6P5	213	−1.02	1.20	−1.40	1.69
889	7.80	70	H12	Fibrinogen alpha chain GN=FGA	P02671-2	165	−1.04	−1.32	1.87	−2.48
890	4.94	16	E3	Actin, alpha 1, skeletal muscle GN=ACTA1	Q5T8M8	124	1.24	1.29	1.31	−1.01
891	5.03	15	E4	Actin, cytoplasmic 2, N-terminal GN=ACTG1	K7EM38	113	1.23	1.14	1.82	−1.59
892	7.57	13	E5	Annexin A2 GN=ANXA2	H0YKV8	109	−1.32	1.17	−1.49	1.75
897	4.30	14	B6	Actin, cytoplasmic 1 GN=ACTB	P60709	242	1.15	1.33	1.51	−1.14
901	8.64	13	L7	Hemoglobin subunit beta GN=HBB	P68871	254	1.17	−1.04	1.56	−1.62
902	8.60	13	L8	Hemoglobin subunit beta GN=HBB	P68871	350	1.02	−1.04	1.61	−1.67
903	8.60	13	L9	Hemoglobin subunit beta GN=HBB	P68871	353	1.18	1.02	1.95	−1.90
904	8.26	13	L15	Hemoglobin subunit beta GN=HBB	P68871	349	1.07	−1.02	1.72	−1.76
905	8.29	13	K23	Hemoglobin subunit beta GN=HBB	P68871	330	−1.09	−1.07	1.59	−1.70
911	4.69	10	I19	SH3 domain-binding glutamic acid-rich protein 3 GN=SH3BGRL3	Q9H299	354	1.39	−1.20	1.59	−1.91

The PBMC protein samples from normal healthy (NH, ) and heart failure (HF, ) subjects were incubated with (Asc⁺) or without (Asc⁻) ascorbate and resolved by 2D-GE approach. Gels were labeled with BODIPY FL N-(2-aminoethyl) maleimide, images were analyzed with SameSpotst™ software, and normalized spot volumes were used for comparison. Proteins spots with ≥|1.5| fold change in abundance or S-nitrosylation level () in HF subjects were subjected to MALDI-TOF MS/MS analysis and those identified with high confidence (score >62) are shown with . Some of the protein spots were also identified by LC MS/MS and this information is presented in brackets in protein and gene name and ID score columns.
¹Ratiometric calculation from BODIPY-fluorescence units in Asc⁺ aliquots (normal versus experimental) was conducted for quantifying the differential abundance of protein spots (Δprotein abundance = Asc⁺ HF/Asc⁺ NH).
²The S-NO modification levels were quantified by calculation of the ratio of fluorescence units from Asc⁻ aliquots (ΔS-NO = Asc⁻ HF/Asc⁻ NH).
³The ratio of ratios, that is, ΔS-NO/Δprotein abundance = [Asc⁻ HF/Asc⁻ NH]/[Asc⁺ HF/Asc⁺ NH], was calculated to obtain the change in S-NO levels normalized for protein abundance. As S-NO modification inhibits the Cys-BODIPY fluorescence, a negative (green) and a positive (red) RoR value would indicate an increase and decrease in S-NO levels, respectively, in HF (versus NH) subjects.