Table 1: Changes in abundance and S-nitrosylation proteome profile of PBMC proteins in human heart failure.

2D-GE dataMALDI MS data2ASC/ASC+ (ΔSNO) ratio1Abundance (ASC+)3S-NO ROR
Spot numberpIMW (kD)Plate numberProtein and gene nameAccession numberMS (LC MS/MS) ID protein scoreNHHFHF versus NHHF versus NH

526.39108F1Vinculin GN=VCLP182067091.14−1.03−1.881.83
546.57108H14Vinculin GN=VCLP18206-211801.081.09−3.293.58
576.68107H15Vinculin GN=VCLP18206-21130−1.03−1.06−3.092.91
586.80107F20Vinculin GN=VCLP18206-21210−1.011.04−3.613.76
596.94107F21Vinculin GN=VCLP1820611701.011.04−3.273.39
607.09105C14FAM212B GN=FAM212BQ9NTI7291.04−1.06−2.382.25
637.53997Vinculin GN=VCLP182062361.12−1.24−1.541.24
796.4896F22Albumin GN=ALBP02768190−1.28−1.14−2.031.78
816.6294F23Keratin, type I cytoskeletal 9 GN=KRT9P35527188−1.18−1.23−2.371.93
926.9387F24Filamin-A GN=FLNAQ5HY54384−1.17−1.56−1.20−1.31
956.8187G2Filamin-A GN=FLNAQ5HY54399−1.19−1.22−2.021.66
994.5484G4Glucose-regulated 78 kDa protein GN=HSPA5P110218491.011.29−1.612.08
1026.6883G5Filamin-A GN=FLNAQ5HY54210−1.26−1.32−1.641.24
1036.4182H21Filamin-A GN=FLNAP21333238−1.28−1.03−1.681.63
1046.5282I20Gelsolin GN=GSNP06396-2182−1.32−1.11−1.721.55
1245.2168G6Heat shock 71 kDa protein GN=HSPA8P11142-23251.04−1.01−1.521.51
1279.0968G7Fibrinogen alpha chain GN=FGAP02671-2621.07−1.651.54−2.53
1315.3568G8Keratin, type I cytoskeletal 10 GN=KRT10P13645296−1.15−1.08−1.721.59
1368.8468E6Transketolase GN=TKTB4E022189−1.03−1.881.84−3.46
1415.97631Serum albumin GN=ALBH0YA55167−1.141.091.071.03
1424.5163G9Tropomyosin 3 GN=TPM3Q5VU597431.011.34−1.832.46
1456.4160A1Serum albumin GN=ALBH0YA55245−1.59−1.28−1.04−1.24
1466.7860C15Serum albumin GN=ALBP02768116−1.00−1.421.32−1.88
1545.3459H24Actin, cytoplasmic 1 GN=ACTBP60709391−1.43−1.07−1.551.44
1805.1454J11Actin, cytoplasmic 1 GN=ACTBP60709353−1.141.18−1.762.07
1855.5053K19Actin, cytoplasmic 1 GN=ACTBP607093651.28−1.161.41−1.63
1977.4851G12Fibrinogen beta chain GN=FGBP02675157−1.14−1.07−1.531.43
2044.2950G13Nucleosome assembly protein 1 GN=NAP1L1F8VRJ2791.21−1.07−1.741.62
2058.5050C16Fibrinogen beta chain GN=FGBP02675336−1.11−1.181.00−1.18
2224.7147J13ATP synthase subunit beta GN=ATP5BH0YH81302−1.061.20−1.922.30
2418.7945J1Transcription factor 4 GN=TCF4 (integrin-linked protein kinase GN=ILK)H3BME836 (305)−1.73−1.16−1.681.45
2616.5741101POTE ankyrin domain family member FA5A3E0121−1.471.16−1.351.57
2627.6841I5Elongation factor Tu, mitochondrial GN=TUFMP49411195−1.52−1.04−1.581.52
2644.8641J18Actin, cytoplasmic 1 GN=ACTBP60709336−1.051.06−1.491.58
2676.414116Actin, cytoplasmic 2, N-terminalF5H0N0106−1.191.33−1.151.53
2728.7041B9Myeloperoxidase GN=MPOP05164-283−1.30−1.471.22−1.79
3034.5438B10Actin, cytoplasmic 1 GN=ACTBP60709911.171.18−1.321.55
3297.1735I10DnaJ homolog subfamily B 11 GN=DNAJB11H7C2Y541−1.25−1.461.17−1.70
3355.5535D6Actin, cytoplasmic 1 GN=ACTBP60709225−1.14−1.091.88−2.04
3375.8134D7Actin, cytoplasmic 1 GN=ACTBP60709456−1.20−1.061.64−1.74
3396.3334105Tubulin beta chain GN=TUBBQ5JP53121−1.28−1.381.19−1.64
3436.7234B13Actin, cytoplasmic 1 GN=ACTBP6070997−1.17−1.361.26−1.71
3443.6333A2Stromal interaction molecule 2 GN=STIM2Q9P246331.43−1.181.98−2.34
3487.1133D8Phosphoglycerate kinase GN=PGK1B7Z7A954−1.23−1.921.49−2.86
3584.5631G24Tropomyosin 1 alpha isoform 7 GN=TPM1D9YZV85151.201.13−1.521.72
3737.3230H1Annexin A1 GN=ANXA1P04083200−1.18−2.15−1.13−1.91
3844.0229L10LVV-hemorphin-7 GN=HBBF8W6P547−1.091.57−1.983.12
3855.7229102Vimentin GN=VIMF5H288171−1.22−1.361.20−1.63
3956.8527E8Keratin, type II cytoskeletal 2 GN=KRT2P35908381−1.46−1.651.70−2.80
3974.0127J23Tropomyosin alpha-4 chain GN=TPM4P679361011.071.41−1.582.22
4006.7026L2Thrombospondin-1 GN=THBS1P07996769−1.141.052.00−1.91
4047.512617Actin, cytoplasmic 1, N-terminal GN=ACTBB4DW521331.23−1.091.91−2.08
4068.0826K1Uncharacterized protein FLJ46347Q6ZRH936−1.07−1.161.88−2.18
4117.98265Myosin-IXa GN=MYO9AH3BMM1461.09−1.272.26−2.86
4244.6925F15Tropomyosin alpha-4 chain GN=TPM4P679369261.731.181.85−1.57
4258.632512Peptidyl-prolyl cis-trans isomerase A GN=PPIAP62937110−1.36−1.171.46−1.71
4316.7024H3Haloacid dehalogenase-like hydrolase domain-containing protein 2 GN=HDHD2K7ER15102−1.20−1.271.42−1.80
4519.3323F16Glyceraldehyde-3-phosphate dehydrogenase GN=GAPDHE7EUT4711.02−1.361.12−1.52
4616.9822B17Annexin A1 GN=ANXA1P040833441.23−1.071.55−1.65
4764.7421D10Actin, cytoplasmic 1 GN=ACTBP607092261.051.221.51−1.24
4918.5820B18Thrombospondin-1 GN=THBS1P079961381.20−1.202.45−2.95
5016.7820H6Peroxiredoxin-6 GN=PRDX6P30041237−1.08−1.481.51−2.23
5058.2220I14Thrombospondin-1 GN=THBS1P079961971.061.071.52−1.42
5076.6120H7Growth factor receptor-bound protein 2 GN=GRB2P62993-280−1.01−1.141.48−1.68
5098.7220L3Thrombospondin-1 GN=THBS1P07996418−1.03−1.292.74−3.53
5117.3020D14Carnitine O-palmitoyltransferase 1 GN=CPT1AP5041632−1.15−1.031.65−1.70
5144.1020E9Transcriptional repressor protein YY1 GN=YY1H0YJV7471.011.27−1.431.82
5155.2619L4Apolipoprotein A-I GN=APOA1P026473521.061.111.56−1.40
5184.2919C21Vimentin GN=VIMB0YJC42031.131.37−1.171.60
5249.3419108Keratin, type I cytoskeletal 10 GN=KRT10P13645286−1.02−1.471.42−2.08
5308.6119L11Keratin, type II cytoskeletal 1 GN=KRT1P04264551.42−1.353.00−4.04
5496.9419J5Glutathione GN=GSH (Ras-related protein Rab-14 GN=RAB14)P0720371 (288)1.10−1.131.57−1.78
5635.9918109Actin, cytoplasmic 2, N-terminal GN=ACTBI3L1U9137−1.531.07−1.601.71
5676.9318C22Glutathione S-transferase P GN=GSTP1P09211286−1.02−1.301.26−1.64
5683.6218E10Transcriptional repressor protein YY1 GN=YY1H0YJV7391.47−1.221.83−2.24
5744.6517C23Tubulin beta-1 chain GN=TUBB1Q9H4B7391.211.28−2.092.66
5883.8817A8Alpha-actinin-1 GN=ACTN1 (Alpha actinin 1 GN=ACTN1)H0YJ1150 (222)1.441.24−1.281.59
5917.5517E12Heat shock 70 kDa protein 1A/1B GN=HSPA1AE7EP11431.01−1.071.43−1.52
5924.3817106Myosin regulatory light chain 12B GN=MYL12BO149503201.431.62−1.712.78
5934.4717C24Actin, cytoplasmic 1 GN=ACTBP60709651.631.75−1.662.91
5958.2717A9Protein S100-A8 GN=S100A8P05109601.59−1.071.65−1.76
6034.7616A10Annexin A5 GN=ANXA5D6RBL5111−1.20−1.371.13−1.55
6055.6416110ATP synthase subunit alpha GN=ATP5A1A8K09295−1.03−1.00−1.021.01
6076.7116B20Actin-related protein 2/3 complex subunit 3 GN=ARPC3O15145501.061.141.64−1.43
6124.1916K21Myosin regulatory light chain 9 GN=MYL9P248442331.191.381.62−1.17
6134.6116D17Actin, cytoplasmic 2 GN=ACTG1P63261611.451.211.78−1.47
6235.1616A12Actin, cytoplasmic 2, N-terminal GN=ACTG1K7EM381301.301.291.87−1.45
6275.47153Annexin A6 GN=ANXA6E5RIU8103−1.251.031.30−1.26
6283.7915E14Myosin light polypeptide 6 GN=MYL6F8VZV53021.301.321.121.18
6294.3515A13Actin, cytoplasmic 1 GN=ACTBP60709651.241.321.151.14
6308.0515K6Peptidyl-prolyl cis-trans isomerase A GN=PPIAP62937199−1.04−1.18−1.10−1.07
6329.0215I15Peptidyl-prolyl cis-trans isomerase A GN=PPIAP62937235−1.01−1.12−1.201.07
6405.121513Actin, cytoplasmic 1, N-terminal GN=ACTBG5E9R01931.451.401.82−1.30
6424.2115E15Actin, cytoplasmic 1 GN=ACTBP607091391.051.25−1.311.64
6447.711521Keratin, type I cytoskeletal 10 GN=KRT10P13645471.161.32−1.071.41
6463.9815H9Myosin light polypeptide 6 GN=MYL6F8VZV53691.161.41−1.391.97
6488.3715D1Bestrophin-3 GN=BEST3 (dermcidin GN=DCD)A8MVM342 (160)1.171.981.291.53
6574.7915A14Actin, cytoplasmic 1 GN=ACTBP607091081.251.121.62−1.44
6624.6514A16Actin, cytoplasmic 1 GN=ACTBP607092631.551.621.65−1.02
6647.0114D19Actin, cytoplasmic 2, N-terminal GN=ACTG1K7EM38411.531.091.29−1.18
6654.4514A17Actin, cytoplasmic 1 GN=ACTBP607092331.561.161.51−1.30
6663.8214B22Mitochondrial carrier homolog 1 GN=MTCH1Q9NZJ7-3321.341.40−1.011.42
6718.6614B23Bestrophin-3 GN=BEST3A8MVM3401.211.432.00−1.40
6734.0314B24Isoform H14 of Myeloperoxidase GN=MPOP05164-2288−1.161.18−1.481.76
6835.0113A22Histone H4 GN=HIST1H4AP628052421.01−1.091.44−1.56
6907.9213L13Hemoglobin subunit beta GN=HBBP68871384−1.15−1.121.50−1.68
7037.7212D3LVV-hemorphin-7 (fragment) GN=HBBF8W6P541−1.131.09−1.491.63
7066.7012E16Protein S100-A11 GN=S100A11P31949260−1.391.27−1.752.22
7184.5311C4Ras-related protein Ral-B GN=RALB (actin cytoplasmic 2 GN=ACTG1)F8WEQ648 (155)1.20−1.301.64−2.14
7324.7211C6Thrombospondin-1 GN=THBS1P079961151.61−2.022.50−5.05
7374.0010D20ATP synthase subunit alpha GN=ATP5A1K7EQT2173−1.16−1.902.40−4.57
7383.8810D21Keratin, type II cytoskeletal 1 GN=KRT1P04264961.07−1.461.85−2.70
7404.860C7Urea transporter 1 GN=SLC14A1K7EJ54391.33−1.351.78−2.40
7444.40023Ras-related protein 1b GN=RAP1BB4DQI858−1.02−1.301.81−2.35
7594.8215D22Actin, cytoplasmic 2, N-terminal GN=ACTG1K7EM382631.271.371.73−1.27
7614.8216D23Keratin, type II cytoskeletal 1 GN=KRT1P042647891.241.311.62−1.24
7624.9116L14Actin, cytoplasmic 2 GN=ACTG1P632612211.341.181.69−1.44
7635.7320A24Actin, cytoplasmic 1 GN=ACTBP60709416−1.06−1.091.89−2.05
7695.3814B1Actin, cytoplasmic 1 GN=ACTBC9JUM1441.07−1.051.59−1.67
7725.3713B2Histone H4 GN=HIST1H4AP62805193−1.36−1.361.13−1.54
7743.7216J7Calmodulin GN=CALM1P62158367−1.111.47−1.512.23
7794.6328D24Actin, cytoplasmic 1 GN=ACTBP607094281.161.21−1.281.55
7814.6127E1Actin, cytoplasmic 1 GN=ACTBP607093421.041.01−1.071.08
7965.2913E19Keratin, type II cytoskeletal 1 GN=KRT1P042641271.29−1.702.35−4.00
7975.3413E20Keratin, type II cuticular Hb3 GN=KRT83P783851651.08−1.111.59−1.77
8046.9610E21LVV-hemorphin-7 (fragment) GN=HBBF8W6P51221.261.36−1.401.90
8088.1111L6Platelet basic protein GN=PPBPP02775901.431.19−1.381.64
8258.6572C8Lactotransferrin delta GN=LTFP02788-2147−1.44−1.651.51−2.49
8548.7467C9Kaliocin-1 GN=LTFE7EQB246−1.09−1.501.22−1.83
8664.8810C10S100-A6 protein GN=S100A6P06703391.46−1.121.94−2.18
8674.910C11LVV-hemorphin-7 GN=HBBF8W6P5651.261.161.50−1.29
8683.5916I18Calmodulin GN=CALM1P62158343−1.011.27−1.702.16
8703.6416K10Calmodulin GN=CALM1 (calmodulin GN=CALM1)E7ETZ063 (215)1.451.60−1.342.15
8713.6916K11Calmodulin GN=CALM1E7ETZ01951.331.31−1.491.95
8773.8055B4Stromal interaction molecule 2 GN=STIM2Q9P246391.31−1.301.69−2.20
8796.8711E23LVV-hemorphin-7 GN=HBBF8W6P5213−1.021.20−1.401.69
8897.8070H12Fibrinogen alpha chain GN=FGAP02671-2165−1.04−1.321.87−2.48
8904.9416E3Actin, alpha 1, skeletal muscle GN=ACTA1Q5T8M81241.241.291.31−1.01
8915.0315E4Actin, cytoplasmic 2, N-terminal GN=ACTG1K7EM381131.231.141.82−1.59
8927.5713E5Annexin A2 GN=ANXA2H0YKV8109−1.321.17−1.491.75
8974.3014B6Actin, cytoplasmic 1 GN=ACTBP607092421.151.331.51−1.14
9018.6413L7Hemoglobin subunit beta GN=HBBP688712541.17−1.041.56−1.62
9028.6013L8Hemoglobin subunit beta GN=HBBP688713501.02−1.041.61−1.67
9038.6013L9Hemoglobin subunit beta GN=HBBP688713531.181.021.95−1.90
9048.2613L15Hemoglobin subunit beta GN=HBBP688713491.07−1.021.72−1.76
9058.2913K23Hemoglobin subunit beta GN=HBBP68871330−1.09−1.071.59−1.70
9114.6910I19SH3 domain-binding glutamic acid-rich protein 3 GN=SH3BGRL3Q9H2993541.39−1.201.59−1.91

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.