Edited by: Thomas Dandekar, Universität Würzburg, Germany
Reviewed by: Claudia M. Calvet Alvarez, University of California, San Diego, United States; Izabela Marques Dourado Bastos, Universidade de Brasília, Brazil; Igor C. Almeida, The University of Texas at El Paso, United States
This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
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Chagas disease (ChD), a neglected parasitic disease recognized as one of the top public health concern in the world, is endemic in Latin America and Mexico (Bonney,
The protein S-nitrosylation (SNO) is an ubiquitous, redox reversible, post-translational modification of cysteine residues that occurs in presence of excessive oxidative and nitrosative stress (Htet Hlaing and Clement,
We previously reported that peripheral blood mononuclear cells (PBMC) carry differential protein abundance signatures of ChD (Garg et al.,
In this study, we report the cysteine SNO fingerprint associated with Chagas disease development. For this, enrolled volunteers were evaluated by cardiologists and grouped according to the severity of their cardiac anomalies following the modified Kuschnir classification criteria based on physical exam, electrocardiography, and transthoracic echocardiography (Sanchez-Montalva et al.,
The institutional review board at the University of Texas Medical Branch at Galveston (IRB 04-257), the ethics committee of the School of Health Sciences at Universidad Nacional de Salta, and the institutional review board of Health Ministry of Salta, Argentina reviewed the study protocol, and all studies were carried out after approval of the human subjects study protocol by the three entities. A written informed consent was obtained from the individuals invited to participate before collection of blood samples. The study enrolled volunteers that came to seek medical assistance at the public hospitals in Salta city, Salta, Argentina. All samples were coded and de-identified before being provided for research purposes.
Sera samples from all enrolled volunteers were analyzed for anti-
We enrolled ChD CA (
The PBMC pellets were lysed in urea buffer (50 mM Tris pH 7.5 containing 7 M urea, 2 M thiourea, and 2% CHAPS), and all PBMC lysates were analyzed by Lowry method to evaluate the protein concentrations, and by L8800 amino acid analyzer (Hitachi High Technologies, Pleasanton, CA) to determine the cysteine (cysteic acid) levels (Koo et al.,
The BD-labeled, Asc+ and Asc− PBMC lysates (100-μg each) were separated by 2D-GE, employing an IPGphor multiple sample isoelectric focusing (IEF) device (GE Healthcare, Chicago, IL) in 1st dimension, and the Criterion Dodeca cell (Bio-Rad, Hercules, CA) in 2nd dimension, following the protocol previously described by us (Garg et al.,
All gels were fixed in 20% methanol/7% acetic acid/10% acetonitrile for 1 h, washed with 20% ethanol / 10% acetonitrile overnight, washed with dH2O, and BD-labeled proteins were imaged at 100 μm resolution (Ex488nm/Em520nm) by using the Typhoon Trio Variable Mode Imager (GE Healthcare). The voltage was set to result in 85–99% of the saturation level for the most abundant protein on the gel. Gel images taken under the BD-specific filters were used to obtain the spot-specific data (Wiktorowicz et al.,
A reference gel from the set of Asc+ gels was selected by Totallabs Ltd SameSpots software (Newcastle, UK). The reference gel was stained overnight with Sypro Ruby Stain (Life Technologies), and imaged at Ex488nm/Em560LPnm. The reference gel image (100 μM resolution) was used to ensure the detection of all protein spots irrespective of presence or absence of cysteine residues and to define the spot boundaries. The matching spot volumes from BD-stained Asc+ and Asc− gels were used to obtain the quantitative spot data (Wiktorowicz et al.,
In total, 166 BODIPY-stained 2DE gels [two gels with either Asc+ or Asc− protein lysates for each sample from the NH (
The SNO modification levels per protein spot from each cohort (NH, ChD CA, ChD CS) were quantified by calculation of the ratio of fluorescence units from Asc− and Asc+ signal for each spot (SNO signal/total protein signal = Asc−/Asc+) (Wiktorowicz et al.,
The final calculation of the SNO modification per unit of protein was carried out according to the following formula, called Ratio of ratios (RoR):
By adjusting for abundance changes, the RoR is used to establish true SNO-specific changes in experimental sample with respect to controls. Moreover, it is important to note that since SNO modification prevents the Cys-BODIPY labeling, a negative value indicates an increase in SNO level (and vice versa) in the sample (Wiktorowicz et al.,
The protein spots that were identified to be differentially abundant and/or differentially SNO-modified (
The MS and MS/MS spectral data were searched against the UniProt human protein database (35,208,664 residues; 87,656 sequences; last accessed: July 12, 2016) by using AB Sciex GPS Explorer (v.3.6) software in conjunction with MASCOT (v.2.2.07, Matrix Science, London, UK) (Garg et al.,
A new set of PBMC samples from NH, ChD CA, and ChD CS subjects (
Sandwich ELISA kits were used to quantify the changes in abundance of actin gamma (Reddot Biotech, Kelowna, Canada; detection range: 0.015–10 ng/mL) and filamin A (LifeSpan Biosciences, Seattle WA; detection range: 1.88–120 ng/mL) polypeptides by following manufacturer's instructions. In brief, aliquot A of PBMC lysates (2 μg in 100 μL/well) were loaded onto 96-well plates pre-coated with target-specific antibody. After 2 h incubation at 4°C, plates were aspirated and sequentially incubated with biotin-conjugated anti-target 2nd antibody (1: 100 dilution), avidin-conjugated horseradish peroxidase (HRP) (1: 100 dilution). The plates were washed between each reagent addition, color was developed with TMB substrate, and the change in absorbance was recorded at 450 nm by using a spectrophotometer (Spectramax 190, Molecular Devices, Sunnyvale, CA). A standard curve was prepared by using recombinant proteins and target protein concentration was plotted as pg per μg total protein (Koo et al.,
The SNO modification levels of actin gamma and filamin A were assessed in aliquot B of each sample by the biotin switch assay. Briefly, free SH (thiol) groups in each sample were blocked with methyl methanethiosulfonate (MMTS), and then protein SNO bonds present in the sample were converted to thiols via transnitrosation with ascorbate. The newly formed SH groups were then labeled by S-biotinylation with biotin-HPDP by using a biotin switch-based, S-Nitrosylated Protein Detection Assay Kit (Cayman Chemicals) following the instructions provided by the manufacturer. Then, 96-well plates pre-coated with antibodies against actin gamma or filamin A were incubated for 2 h with biotin-derivatized protein lysates (2 μg in 100 μL/well). Plates were washed to remove the unbound proteins and then incubated for 10 min at room temperature with the avidin-conjugated HRP (1:3,000 dilution, BioLegend, San Diego, CA). The TMB substrate was added, and the change in absorbance reflecting the levels of biotin-bound SNO-modified actin gamma or filamin A was measured by spectrophotometry (Koo et al.,
MARS is a non-parametric regression procedure that automatically models non-linearities and interactions between variables (Friedman and Roosen,
To assess the biological meaning of the proteome datasets, we used the IPA web-based application (Ingenuity Systems, Redwood city, CA) (Thomas and Bonchev,
A scheme of the steps involved in SNO proteomic analysis is presented in Figure
Schematic of work flow. Human PBMCs were obtained from volunteers categorized as seropositive, Chagas disease (ChD), clinically asymptomatic (ChD CA,
Representative two-dimensional gel images of protein spots in PBMC of Chagas disease subjects. BD-labeled PBMC lysates were separated in 1st dimension by isoelectric focusing on 11 cm non-linear pH 3–11 immobilized pH gradient strips, and in 2nd dimension by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on an 8–16% gradient gel. Gel images were obtained at 100 μm resolution to quantify BD-labeled proteins (Ex488nm/Em520 ± 15nm). Shown are the representative gel images of Asc+
Of the 312 protein spots submitted to mass spectrometry, 249 protein/peptide spots were correctly identified (Table
Detailed S-Nitrosylation proteome profile of PBMC proteins in human Chagas disease
52 | 6.4 | 108 | Isoform 1 of Vinculin = VCL | 709 | 1.14 | −1.11 | −1.05 | −1.27 | −1.19 | 1.06 | |
54 | 6.6 | 108 | Isoform 1 of Vinculin = VCL | 1,180 | 1.08 | −1.27 | 1.15 | −1.37 | 1.06 | 1.46 | |
57 | 6.7 | 107 | Isoform 1 of Vinculin = VCL | 1,130 | −1.03 | −1.10 | 1.15 | −1.07 | 1.19 | 1.26 | |
58 | 6.8 | 107 | Isoform 1 of Vinculin = VCL | 1,210 | −1.01 | −1.05 | 1.03 | −1.05 | 1.04 | 1.09 | |
59 | 6.9 | 107 | Vinculin = VCL | 1,170 | 1.01 | 1.03 | 1.06 | 1.02 | 1.05 | 1.03 | |
60 | 7.1 | 105 | PAK4-inhibitor INKA2 = FAM212B | 29 | 1.04 | 1.17 | 1.20 | 1.13 | 1.16 | 1.03 | |
63 | 7.5 | 99 | Vinculin = VCL | 236 | 1.12 | 1.18 | −1.10 | 1.06 | −1.23 | −1.30 | |
69 | 5.4 | 99 | Actin, cytoplasmic 1 = ACTB | 347 | −1.36 | −1.09 | −1.11 | 1.24 | 1.23 | −1.01 | |
79 | 6.5 | 96 | Serum albumin = ALB | 190 | −1.28 | 1.09 | 1.03 | 1.39 | 1.32 | −1.05 | |
81 | 6.6 | 94 | Keratin, type I cytoskeletal 9 = KRT9 | 188 | −1.18 | 1.10 | −1.02 | 1.29 | 1.15 | −1.12 | |
89 | 5.6 | 88 | Isoform 1 of Vinculin = VCL | 323 | 1.08 | −1.11 | 1.09 | −1.19 | 1.02 | 1.21 | |
90 | 5.7 | 87 | POTE ankyrin domain family member E = POTEE | 135 | −1.04 | −1.01 | 1.03 | 1.03 | 1.07 | 1.05 | |
91 | 5.8 | 87 | Filamin-A = FLNA | 330 | −1.13 | 1.02 | −1.09 | 1.15 | 1.04 | −1.11 | |
92 | 6.9 | 87 | Filamin-A = FLNA | 384 | −1.17 | 1.18 | −1.09 | 1.38 | 1.07 | −1.29 | |
93 | 5.9 | 87 | Filamin-A = FLNA | 389 | −1.21 | 1.06 | −1.04 | 1.27 | 1.16 | −1.10 | |
94 | 6.0 | 87 | Filamin-A = FLNA | 238 | −1.24 | 1.06 | −1.04 | 1.32 | 1.20 | −1.11 | |
95 | 6.8 | 87 | Filamin-A = FLNA | 399 | −1.19 | 1.16 | −1.22 | 1.38 | −1.02 | −1.42 | |
96 | 6.1 | 86 | Filamin-A = FLNA | 169 | −1.21 | −1.12 | −1.01 | 1.08 | 1.20 | 1.11 | |
97 | 6.2 | 85 | Filamin-A = FLNA | 164 | −1.26 | −1.28 | −1.18 | −1.02 | 1.06 | 1.08 | |
99 | 4.5 | 84 | 78 kDa glucose-regulated protein GRP78 = HSPA5 | 849 | 1.01 | 1.03 | 1.05 | 1.02 | 1.04 | 1.01 | |
100 | 6.3 | 83 | Filamin-A = FLNA | 325 | −1.28 | −1.24 | −1.12 | 1.03 | 1.14 | 1.11 | |
101 | 5.4 | 83 | Actin, cytoplasmic 1 = ACTB | 297 | −1.36 | −1.15 | −1.07 | 1.18 | 1.28 | 1.08 | |
102 | 6.7 | 83 | Filamin-A = FLNA | 210 | −1.26 | 1.41 | −1.06 | 1.79 | 1.19 | −1.50 | |
103 | 6.4 | 82 | Filamin-A = FLNA | 238 | −1.28 | 1.15 | −1.08 | 1.47 | 1.18 | −1.24 | |
104 | 6.5 | 82 | Isoform 2 of Gelsolin = GSN | 182 | −1.32 | 1.18 | 1.04 | 1.56 | 1.37 | −1.14 | |
118 | 5.6 | 69 | Actin, cytoplasmic 2 = ACTG1 | 269 | −1.04 | −1.15 | −1.10 | −1.11 | −1.06 | 1.05 | |
121 | 5.4 | 68 | Actin alpha 1, skeletal = ACTA1 | 227 | −1.32 | −1.04 | −1.11 | 1.27 | 1.18 | −1.07 | |
124 | 5.2 | 68 | Isoform 2 of Heat shock cognate 71 kDa protein = HSPA8 | 325 | 1.04 | −1.20 | −1.13 | −1.25 | −1.18 | 1.06 | |
127 | 9.1 | 68 | Isoform 2 of Fibrinogen alpha = FGA | 62 | 1.07 | 1.07 | −1.22 | 1.00 | −1.30 | −1.31 | |
131 | 5.4 | 68 | Keratin, type I cytoskeletal 10 = KRT10 | 296 | −1.15 | 1.01 | −1.04 | 1.16 | 1.11 | −1.05 | |
136 | 8.8 | 68 | Transketolase = TKT | 189 | −1.03 | −1.15 | −1.20 | −1.11 | −1.17 | −1.05 | |
141 | 6.0 | 63 | Serum albumin = ALB | 167 | −1.14 | −1.52 | 1.13 | −1.33 | 1.29 | 1.72 | |
142 | 4.5 | 63 | Tropomyosin 3 = TPM3 | 743 | 1.01 | −1.01 | 1.04 | −1.02 | 1.03 | 1.06 | |
145 | 6.4 | 60 | Serum albumin = ALB | 245 | −1.59 | −1.14 | −1.16 | 1.40 | 1.37 | −1.02 | |
146 | 6.8 | 60 | Serum albumin = ALB | 116 | −1.00 | 1.09 | −1.08 | 1.09 | −1.07 | −1.17 | |
147 | 7.0 | 60 | Early endosome antigen 1 = EEA1 | 28 | −1.16 | 1.02 | −1.07 | 1.18 | 1.08 | −1.10 | |
148 | 7.4 | 60 | Keratin, type I cytoskeletal 9 = KRT9 | 182 | −1.44 | 1.02 | −1.05 | 1.48 | 1.37 | −1.08 | |
149 | 7.1 | 60 | Transcriptional repressor protein YY1 fragment = YY1 | 42 | −1.14 | −1.06 | 1.04 | 1.08 | 1.19 | 1.11 | |
154 | 5.3 | 59 | Actin, cytoplasmic 1 = ACTB | 391 | −1.43 | 1.10 | −1.03 | 1.58 | 1.38 | −1.14 | |
165 | 4.2 | 55 | Isoform 3 of Integrin alpha-IIb = ITGA2B | 268 | −1.09 | 1.05 | 1.02 | 1.15 | 1.11 | −1.03 | |
167 | 8.4 | 55 | Pyruvate kinase = PKM | 197 | 1.01 | −1.01 | 1.09 | −1.02 | 1.07 | 1.10 | |
168 | 8.6 | 55 | Myeloperoxidase = MPO | 107 | −1.33 | 1.03 | 1.05 | 1.37 | 1.40 | 1.02 | |
169 | 8.8 | 55 | Pyruvate kinase isozymes M1/M2 = PKM | 371 | −1.07 | 1.07 | 1.21 | 1.15 | 1.29 | 1.13 | |
170 | 9.4 | 55 | Isoform H7 of Myeloperoxidase = MPO | 79 | 1.14 | 1.48 | −1.15 | 1.31 | −1.30 | −1.70 | |
180 | 5.1 | 54 | Actin, cytoplasmic 1 = ACTB | 353 | −1.14 | 1.17 | −1.12 | 1.33 | 1.01 | −1.32 | |
184 | 5.3 | 53 | Actin, cytoplasmic 1 = ACTB | 394 | −1.32 | −1.05 | −1.09 | 1.25 | 1.21 | −1.04 | |
185 | 5.5 | 53 | Actin, cytoplasmic 1 = ACTB | 365 | 1.28 | −1.29 | −1.03 | −1.65 | −1.32 | 1.26 | |
186 | 5.7 | 53 | Actin, cytoplasmic 1 = ACTB | 333 | 1.12 | −1.21 | −1.36 | −1.36 | −1.52 | −1.12 | |
191 | 6.9 | 52 | Fibrinogen beta = FGB | 107 | 1.18 | 1.12 | −1.46 | −1.05 | −1.72 | −1.64 | |
195 | 7.2 | 51 | Fibrinogen beta = FGB | 163 | 1.07 | 1.01 | 1.01 | −1.06 | −1.05 | 1.00 | |
196 | 7.0 | 51 | Fibrinogen beta = FGB | 170 | 1.06 | 1.02 | −1.19 | −1.04 | −1.26 | −1.21 | |
197 | 7.5 | 51 | Fibrinogen beta = FGB | 157 | −1.14 | −1.05 | −1.10 | 1.09 | 1.04 | −1.05 | |
199 | 7.3 | 50 | Keratin, type I cytoskeletal 10 = KRT10 | 108 | −1.11 | 1.05 | −1.00 | 1.16 | 1.10 | −1.05 | |
200 | 7.8 | 50 | Fibrinogen beta = FGB | 329 | −1.05 | −1.12 | −1.16 | −1.07 | −1.10 | −1.03 | |
201 | 7.6 | 50 | Fibrinogen beta = FGB | 64 | −1.03 | −1.05 | −1.14 | −1.02 | −1.11 | −1.09 | |
202 | 8.1 | 50 | Fibrinogen beta = FGB | 232 | −1.05 | −1.01 | −1.06 | 1.04 | −1.01 | −1.05 | |
204 | 4.3 | 50 | Nucleosome assembly protein 1-like 1 = NAP1L1 | 79 | 1.21 | 1.12 | 1.01 | −1.08 | −1.20 | −1.11 | |
205 | 8.5 | 50 | Fibrinogen beta = FGB | 336 | −1.11 | 1.00 | 1.01 | 1.11 | 1.12 | 1.01 | |
207 | 8.0 | 50 | Cyclin-dependent kinase 4 = CDK4 | 41 | −1.02 | −1.03 | −1.24 | −1.01 | −1.22 | −1.21 | |
208 | 8.7 | 50 | Isoform 2 of Fibrinogen alpha = FGA | 101 | −1.13 | −1.18 | −1.04 | −1.05 | 1.08 | 1.13 | |
213 | 7.7 | 49 | Fibrinogen beta = FGB | 156 | −1.01 | −1.03 | −1.11 | −1.01 | −1.10 | −1.08 | |
214 | 8.1 | 49 | Fibrinogen beta = FGB | 212 | −1.02 | −1.06 | −1.09 | −1.04 | −1.07 | −1.03 | |
219 | 4.9 | 48 | ATP synthase subunit beta = ATP5B | 409 | −1.07 | −1.03 | −1.13 | 1.04 | −1.06 | −1.10 | |
222 | 4.7 | 47 | ATP synthase subunit beta = ATP5B | 302 | −1.06 | 1.03 | −1.31 | 1.09 | −1.24 | −1.35 | |
224 | 8.2 | 47 | Isoform 2 of UTP–glucose-1-phosphate uridylyltransferase = UGP2 | 85 | −1.07 | 1.08 | −1.24 | 1.16 | −1.16 | −1.35 | |
226 | 6.7 | 47 | Alpha-enolase = ENO1 | 167 | 1.11 | 1.03 | 1.01 | −1.08 | −1.10 | −1.02 | |
232 | 5.8 | 45 | Actin, cytoplasmic 1 = ACTB | 281 | −1.12 | −1.21 | −1.07 | −1.07 | 1.05 | 1.13 | |
233 | 7.1 | 45 | Alpha-enolase = ENO1 | 128 | −1.12 | −1.03 | 1.11 | 1.09 | 1.25 | 1.15 | |
234 | 7.2 | 45 | Alpha-enolase = ENO1 | 363 | −1.07 | −1.04 | 1.10 | 1.04 | 1.18 | 1.14 | |
235 | 7.4 | 45 | Enolase = ENO3 | 85 | −1.23 | −1.07 | −1.07 | 1.14 | 1.15 | 1.00 | |
236 | 9.1 | 45 | Vasodilator-stimulated phosphoprotein = VASP | 187 | −1.11 | 1.09 | 1.16 | 1.21 | 1.29 | 1.06 | |
238 | 5.5 | 45 | Actin, cytoplasmic 1 = ACTB | 294 | 1.01 | −1.14 | −1.06 | −1.16 | −1.08 | 1.08 | |
240 | 7.6 | 45 | Alpha-enolase = ENO1 | 347 | −1.14 | −1.05 | −1.06 | 1.09 | 1.08 | −1.01 | |
241 | 8.8 | 45 | Transcription factor 4 = TCF4 | 306 | −1.73 | −1.19 | 1.03 | 1.45 | 1.79 | 1.23 | |
242 | 9.3 | 45 | Isoform 2 of Septin-7 = SEPT7 | 118 | −1.40 | 1.05 | 1.03 | 1.46 | 1.44 | −1.01 | |
243 | 8.7 | 44 | Isoform Delta of Lactotransferrin = LTF | 206 | −1.04 | 1.01 | 1.18 | 1.05 | 1.23 | 1.18 | |
244 | 5.1 | 44 | Actin, cytoplasmic 1 = ACTB | 390 | −1.29 | −1.11 | −1.23 | 1.16 | 1.05 | −1.10 | |
246 | 8.2 | 44 | Acyl-coenzyme A synthetase mitoch = ACSM2B | 39 | −1.17 | −1.07 | 1.28 | 1.10 | 1.50 | 1.36 | |
247 | 8.4 | 44 | Alpha-enolase = ENO1 | 423 | −1.15 | 1.01 | 1.18 | 1.16 | 1.35 | 1.16 | |
254 | 7.3 | 42 | Actin, cytoplasmic 1 = ACTB | 318 | −1.10 | 1.20 | 1.13 | 1.31 | 1.24 | −1.06 | |
255 | 7.5 | 42 | Actin, cytoplasmic 2 = ACTG1 | 55 | −1.36 | 1.06 | −1.03 | 1.44 | 1.32 | −1.09 | |
256 | 7.6 | 42 | Alpha-enolase = ENO1 | 166 | −1.19 | 1.15 | −1.03 | 1.37 | 1.16 | −1.18 | |
257 | 8.0 | 42 | 60 kDa heat shock protein, mitoch = HSPD1 | 51 | −1.30 | 1.08 | 1.01 | 1.40 | 1.32 | −1.06 | |
260 | 7.9 | 41 | Elongation factor Tu, mitoch = TUFM | 338 | −1.41 | 1.20 | −1.00 | 1.70 | 1.41 | −1.20 | |
261 | 6.6 | 41 | POTE ankyrin domain family member F | 121 | −1.47 | 1.22 | −1.10 | 1.79 | 1.33 | −1.34 | |
262 | 7.7 | 41 | Elongation factor Tu, mitoch = TUFM | 195 | −1.52 | 1.16 | −1.18 | 1.76 | 1.29 | −1.37 | |
264 | 4.9 | 41 | Actin, cytoplasmic 1 = ACTB | 336 | −1.05 | −1.01 | −1.07 | 1.03 | −1.02 | −1.06 | |
267 | 6.4 | 41 | Actin, cytoplasmic 2 = ACTG1 | 106 | −1.19 | 1.06 | −1.26 | 1.25 | −1.06 | −1.33 | |
268 | 6.7 | 41 | Actin, cytoplasmic 1 = ACTB | 298 | −1.12 | 1.18 | 1.02 | 1.32 | 1.14 | −1.16 | |
269 | 6.9 | 41 | Actin, cytoplasmic 2 = ACTG1 | 170 | −1.16 | 1.03 | 1.06 | 1.19 | 1.22 | 1.03 | |
270 | 5.0 | 41 | Actin, cytoplasmic 1 = ACTB | 383 | −1.14 | −1.03 | −1.18 | 1.10 | −1.04 | −1.14 | |
271 | 8.6 | 41 | Isoform 2 of Fibrinogen alpha = FGA | 72 | −1.15 | 1.10 | −1.02 | 1.27 | 1.13 | −1.12 | |
272 | 8.7 | 41 | Isoform H14 of Myeloperoxidase = MPO | 83 | −1.30 | −1.16 | −1.13 | 1.12 | 1.15 | 1.02 | |
273 | 8.8 | 41 | Isoform 2 of Fibrinogen alpha = FGA | 54 | −1.18 | −1.07 | −1.03 | 1.10 | 1.15 | 1.04 | |
275 | 5.2 | 40 | Actin, cytoplasmic 1 = ACTB | 418 | −1.26 | −1.21 | −1.15 | 1.04 | 1.10 | 1.05 | |
279 | 7.3 | 39 | UPF0515 protein = C19orf66 | 34 | −1.17 | 1.03 | 1.12 | 1.20 | 1.30 | 1.08 | |
283 | 5.9 | 39 | Actin, cytoplasmic 2 = ACTG1 | 170 | −1.45 | −1.01 | −1.03 | 1.44 | 1.41 | −1.02 | |
296 | 8.1 | 38 | Actin, cytoplasmic 2, N-terminal = ACTG1 | 36 | −1.20 | −1.08 | −1.06 | 1.11 | 1.13 | 1.02 | |
300 | 8.4 | 38 | Phosphoglycerate kinase 1 = PGK1 | 250 | −1.07 | −1.19 | −1.07 | −1.12 | 1.00 | 1.12 | |
303 | 4.5 | 38 | Actin, cytoplasmic 1 = ACTB | 91 | 1.17 | 1.23 | −1.01 | 1.04 | −1.19 | −1.25 | |
304 | 8.7 | 38 | Vimentin = VIM | 479 | −1.00 | −1.15 | −1.20 | −1.15 | −1.20 | −1.04 | |
305 | 9.1 | 38 | Phosphoglycerate kinase 1 = PGK1 | 194 | −1.07 | 1.06 | −1.10 | 1.13 | −1.03 | −1.16 | |
307 | 4.4 | 37 | Vimentin = VIM | 698 | 1.14 | 1.13 | 1.19 | −1.01 | 1.04 | 1.05 | |
309 | 7.0 | 37 | Leukocyte elastase inhibitor = SERPINB1 | 388 | −1.07 | −1.03 | −1.04 | 1.04 | 1.03 | −1.02 | |
310 | 7.1 | 37 | Actin, cytoplasmic 1 = ACTB | 41 | −1.19 | 1.13 | 1.01 | 1.34 | 1.20 | −1.12 | |
312 | 8.7 | 37 | Vimentin = VIM | 632 | 1.03 | −1.07 | 1.00 | −1.10 | −1.03 | 1.07 | |
320 | 5.1 | 36 | Actin, cytoplasmic 1 = ACTB | 307 | 1.16 | −1.37 | −1.07 | −1.59 | −1.25 | 1.28 | |
321 | 5.3 | 36 | Actin, cytoplasmic 1 = ACTB | 247 | −1.07 | −1.42 | −1.02 | −1.32 | 1.05 | 1.39 | |
324 | 4.7 | 36 | Isoform 2 of Tropomyosin beta = TPM2 | 258 | 1.15 | −1.10 | 1.10 | −1.27 | −1.04 | 1.22 | |
329 | 7.2 | 35 | DnaJ homolog subfamily B member 11 = DNAJB11 | 41 | −1.25 | 1.03 | −1.06 | 1.29 | 1.17 | −1.10 | |
330 | 7.3 | 35 | Cyclin-dependent kinase 4 = CDK4 | 39 | −1.16 | −1.11 | 1.07 | 1.05 | 1.24 | 1.18 | |
333 | 7.6 | 35 | Cyclin-dependent kinase 4 = CDK4 | 38 | −1.19 | 1.05 | −1.03 | 1.25 | 1.16 | −1.08 | |
335 | 5.6 | 35 | Actin, cytoplasmic 1 = ACTB | 225 | −1.14 | −1.28 | −1.15 | −1.12 | −1.02 | 1.11 | |
337 | 5.8 | 34 | Actin, cytoplasmic 1 = ACTB | 456 | −1.20 | −1.12 | −1.05 | 1.07 | 1.15 | 1.07 | |
339 | 6.3 | 34 | Tubulin beta chain | 121 | −1.28 | −1.44 | −1.19 | −1.12 | 1.07 | 1.20 | |
343 | 6.7 | 34 | Actin, cytoplasmic 1 = ACTB | 97 | −1.17 | 1.14 | 1.09 | 1.34 | 1.27 | −1.05 | |
344 | 3.6 | 33 | Stromal interaction molecule 2 = STIM2 | 33 | 1.43 | 1.06 | −1.02 | −1.35 | −1.46 | −1.07 | |
346 | 9.0 | 33 | Glyceraldehyde-3-phosphate dehydrogenase = GAPDH | 164 | 1.28 | −1.09 | −1.05 | −1.39 | −1.34 | 1.04 | |
348 | 7.1 | 33 | Phosphoglycerate kinase = PGK1 | 54 | −1.23 | 1.08 | 1.19 | 1.32 | 1.45 | 1.10 | |
353 | 8.7 | 32 | Fructose-bisphosphate aldolase A = ALDOA | 146 | 1.13 | −1.16 | −1.07 | −1.31 | −1.20 | 1.09 | |
355 | 5.2 | 32 | Talin 1 = TLN1 | 187 | −1.13 | 1.00 | 1.27 | 1.14 | 1.44 | 1.26 | |
357 | 8.5 | 32 | Annexin = ANXA2 | 268 | 1.28 | −1.05 | −1.24 | −1.34 | −1.58 | −1.18 | |
358 | 4.6 | 31 | Tropomyosin 1 (Alpha) isoform 7 = TPM1 | 515 | 1.20 | 1.13 | 1.18 | −1.06 | −1.02 | 1.04 | |
364 | 5.0 | 31 | ATP synthase subunit beta = ATP5B | 165 | −1.40 | −1.02 | 1.04 | 1.38 | 1.46 | 1.06 | |
367 | 8.5 | 31 | Glyceraldehyde-3-phosphate dehydrogenase = GAPDH | 82 | 1.33 | −1.10 | 1.09 | −1.46 | −1.22 | 1.19 | |
372 | 8.7 | 31 | Heterogeneous nuclear ribonucleoproteins A2/B1 = HNRNPA2B1 | 226 | 1.02 | −1.09 | 1.14 | −1.11 | 1.11 | 1.23 | |
373 | 7.3 | 30 | Annexin A1 = ANXA1 | 200 | −1.18 | −1.13 | 1.27 | 1.04 | 1.49 | 1.44 | |
377 | 8.7 | 30 | Isoform 2 of Fibrinogen alpha = FGA | 79 | 1.50 | 1.07 | 1.42 | −1.40 | −1.05 | 1.33 | |
379 | 9.3 | 30 | Fructose-bisphosphate aldolase = ALDOA | 176 | −1.40 | −1.26 | −1.15 | 1.12 | 1.22 | 1.10 | |
382 | 4.7 | 30 | Actin, cytoplasmic 1 = ACTB | 468 | 1.08 | −1.10 | 1.05 | −1.19 | −1.03 | 1.16 | |
384 | 4.0 | 29 | LVV-hemorphin-7 = HBB | 47 | −1.09 | −1.20 | −1.03 | −1.10 | 1.07 | 1.17 | |
385 | 5.7 | 29 | Vimentin = VIM | 171 | −1.22 | 1.10 | −1.28 | 1.34 | −1.05 | −1.40 | |
389 | 6.1 | 28 | Annexin A3 = ANXA3 | 549 | −1.20 | −1.22 | 1.10 | −1.01 | 1.33 | 1.34 | |
390 | 8.5 | 28 | Actin-related protein 2/3 complex subunit 2 = ARPC2 | 64 | 1.09 | 1.01 | −1.12 | −1.07 | −1.22 | −1.14 | |
394 | 6.4 | 27 | Actin, cytoplasmic 1 = ACTB | 97 | −1.32 | −1.22 | −1.19 | 1.08 | 1.10 | 1.02 | |
395 | 6.9 | 27 | Keratin, type II cytoskeletal 2 epidermal = KRT2 | 381 | −1.46 | 1.16 | 1.09 | 1.68 | 1.59 | −1.06 | |
397 | 4.0 | 27 | Tropomyosin alpha-4 = TPM4 | 101 | 1.07 | −1.05 | −1.08 | −1.12 | −1.15 | −1.02 | |
400 | 6.7 | 26 | Thrombospondin-1 = THBS1 | 769 | −1.14 | 1.36 | 1.18 | 1.56 | 1.35 | −1.16 | |
403 | 7.2 | 26 | Purine nucleoside phosphorylase = PNP | 48 | −1.01 | 1.04 | 1.07 | 1.05 | 1.08 | 1.03 | |
404 | 7.5 | 26 | Actin, cytoplasmic 1 = ACTB | 133 | 1.23 | 1.16 | −1.09 | −1.05 | −1.34 | −1.27 | |
406 | 8.1 | 26 | Uncharacterized protein FLJ46347 | 36 | −1.07 | −1.12 | −1.12 | −1.04 | −1.05 | −1.00 | |
411 | 8.0 | 26 | Unconventional myosin-IXa = MYO9A | 46 | 1.09 | −1.02 | −1.16 | −1.11 | −1.26 | −1.14 | |
421 | 7.4 | 25 | Thrombospondin-1 = THBS1 | 368 | −1.01 | 1.05 | 1.04 | 1.06 | 1.05 | −1.01 | |
423 | 6.9 | 25 | Keratin, type I cytoskeletal 9 = KRT9 | 48 | 1.06 | 1.14 | 1.13 | 1.08 | 1.07 | −1.01 | |
424 | 4.7 | 25 | Tropomyosin alpha-4 = TPM4 | 926 | 1.73 | 1.03 | 1.05 | −1.68 | −1.65 | 1.01 | |
425 | 8.6 | 25 | Peptidyl-prolyl cis-trans isomerase A = PPIA | 110 | −1.36 | −1.22 | −1.04 | 1.12 | 1.30 | 1.17 | |
426 | 7.6 | 24 | Purine nucleoside phosphorylase = PNP | 154 | 1.42 | −1.01 | −1.02 | −1.43 | −1.44 | −1.01 | |
430 | 6.5 | 24 | Thrombospondin-1 = THBS1 | 860 | −1.10 | 1.01 | 1.25 | 1.11 | 1.37 | 1.24 | |
431 | 6.7 | 24 | Haloacid dehalogenase-like hydrolase domain-containing protein 2 = HDHD2 | 102 | −1.20 | 1.19 | 1.32 | 1.43 | 1.58 | 1.11 | |
438 | 8.8 | 23 | WD repeat-containing protein 49 = WDR49 | 46 | −1.05 | −1.11 | 1.01 | −1.06 | 1.06 | 1.13 | |
441 | 7.1 | 23 | Glutathione S-transferase omega-1 = GSTO1 | 296 | −1.12 | 1.14 | 1.10 | 1.28 | 1.24 | −1.03 | |
451 | 9.3 | 23 | Glyceraldehyde-3-phosphate dehydrogenase = GAPDH | 71 | 1.02 | 1.17 | −1.07 | 1.14 | −1.09 | −1.24 | |
455 | 7.9 | 22 | Ras suppressor protein 1 = RSU1 | 89 | −1.02 | −1.06 | −1.20 | −1.03 | −1.17 | −1.14 | |
461 | 7.0 | 22 | Annexin A1 = ANXA1 | 344 | 1.23 | −1.16 | −1.02 | −1.42 | −1.25 | 1.14 | |
462 | 7.3 | 22 | Rho GTPase-activating protein 39 = ARHGAP39 | 31 | 1.19 | −1.05 | 1.04 | −1.25 | −1.14 | 1.09 | |
465 | 8.4 | 22 | Ras suppressor protein 1 = RSU1 | 204 | 1.17 | −1.06 | −1.01 | −1.24 | −1.17 | 1.06 | |
476 | 4.7 | 21 | Actin, cytoplasmic 1 = ACTB | 226 | 1.05 | −1.04 | −1.07 | −1.09 | −1.13 | −1.04 | |
479 | 5.4 | 21 | Actin, cytoplasmic 1 = ACTB | 334 | 1.13 | −1.14 | −1.12 | −1.29 | −1.26 | 1.02 | |
481 | 6.8 | 20 | Peroxiredoxin-6 = PRDX6 | 306 | −1.36 | −1.09 | 1.03 | 1.25 | 1.40 | 1.12 | |
486 | 7.0 | 20 | Serum albumin = ALB | 165 | −1.45 | 1.11 | 1.06 | 1.61 | 1.54 | −1.05 | |
489 | 7.6 | 20 | Keratin, type I cytoskeletal 10 = KRT10 | 120 | −1.24 | −1.07 | −1.00 | 1.16 | 1.24 | 1.07 | |
491 | 8.6 | 20 | Thrombospondin-1 = THBS1 | 138 | 1.20 | 1.60 | 1.12 | 1.33 | −1.07 | −1.43 | |
494 | 5.0 | 20 | Actin, cytoplasmic 2, N-terminally processed = ACTG1 | 177 | 1.17 | 1.00 | −1.05 | −1.16 | −1.22 | −1.05 | |
495 | 5.1 | 20 | Apolipoprotein A-I = APOA1 | 224 | 1.03 | 1.03 | −1.04 | 1.00 | −1.07 | −1.08 | |
498 | 6.2 | 20 | Actin, cytoplasmic 1 = ACTB | 140 | −1.03 | −1.47 | −1.21 | −1.43 | −1.18 | 1.21 | |
501 | 6.8 | 20 | Peroxiredoxin-6 = PRDX6 | 237 | −1.08 | 1.20 | −1.15 | 1.30 | −1.06 | −1.38 | |
502 | 7.1 | 20 | Keratin, type II cytoskeletal 1 = KRT1 | 153 | −1.05 | 1.13 | −1.10 | 1.19 | −1.05 | −1.25 | |
503 | 7.7 | 20 | Myeloblastin = PRTN3 | 54 | −1.21 | −1.06 | −1.09 | 1.15 | 1.12 | −1.03 | |
505 | 8.2 | 20 | Thrombospondin-1 = THBS1 | 197 | 1.06 | 1.09 | 1.04 | 1.02 | −1.02 | −1.05 | |
506 | 8.9 | 20 | Parathyroid hormone 2 receptor = PTHR2 | 42 | 1.17 | −1.43 | 1.07 | −1.67 | −1.10 | 1.52 | |
507 | 6.6 | 20 | Isoform 2 of Growth factor receptor-bound protein 2 = GRB2 | 80 | −1.01 | 1.15 | −1.11 | 1.16 | −1.10 | −1.27 | |
508 | 7.4 | 20 | Serum albumin = ALB | 224 | 1.12 | 1.02 | −1.03 | −1.10 | −1.15 | −1.05 | |
509 | 8.7 | 20 | Thrombospondin-1 = THBS1 | 418 | −1.03 | −1.01 | −1.04 | 1.02 | −1.00 | −1.02 | |
511 | 7.3 | 20 | Carnitine O-palmitoyl-transferase 1, liver isoform = CPT1A | 32 | −1.15 | −1.17 | −1.05 | −1.02 | 1.09 | 1.11 | |
512 | 5.5 | 20 | Actin, cytoplasmic 2 = ACTG1 | 150 | 1.14 | −1.11 | −1.25 | −1.26 | −1.42 | −1.12 | |
514 | 4.1 | 20 | Transcriptional repressor protein YY1 = YY1 | 47 | 1.01 | 1.01 | −1.09 | −1.00 | −1.10 | −1.10 | |
515 | 5.3 | 19 | Apolipoprotein A-I = APOA1 | 352 | 1.06 | −1.07 | −1.10 | −1.13 | −1.17 | −1.03 | |
518 | 4.3 | 19 | Vimentin = VIM | 203 | 1.13 | 1.14 | 1.22 | 1.01 | 1.08 | 1.07 | |
524 | 9.3 | 19 | Keratin, type I cytoskeletal 10 = KRT10 | 286 | −1.02 | 1.20 | −1.13 | 1.23 | −1.10 | −1.35 | |
530 | 8.6 | 19 | Keratin, type II cytoskeletal 1 = KRT1 | 55 | 1.42 | 1.13 | 1.02 | −1.25 | −1.39 | −1.11 | |
531 | 7.2 | 19 | Proteasome subunit beta type-3 = PSMB3 | 55 | −1.25 | 1.21 | 1.26 | 1.52 | 1.58 | 1.04 | |
533 | 7.1 | 19 | Keratin, type II cytoskeletal 1 = KRT1 | 180 | 1.10 | 1.10 | 1.18 | −1.00 | 1.07 | 1.07 | |
535 | 8.0 | 19 | Proteasome subunit beta type-2 = PSMB2 | 82 | 1.00 | 1.17 | −1.05 | 1.17 | −1.05 | −1.23 | |
540 | 4.3 | 19 | Vimentin = VIM | 182 | 1.19 | 1.16 | 1.20 | −1.03 | 1.01 | 1.04 | |
544 | 8.4 | 19 | Superoxide dismutase = SOD2 | 209 | 1.08 | 1.08 | 1.04 | −1.00 | −1.04 | −1.03 | |
549 | 6.9 | 19 | Glutathione peroxidase 1 = GPX1 | 71 | 1.10 | 1.06 | 1.03 | −1.04 | −1.07 | −1.03 | |
552 | 3.9 | 19 | Isoform 3 of mitoch carrier homolog 1 = MTCH1 | 31 | 1.42 | 1.03 | −1.23 | −1.38 | −1.75 | −1.27 | |
554 | 7.1 | 18 | Isoform 3 of mitoch. carrier homolog 1 = MTCH1 | 38 | 1.27 | 1.38 | 1.53 | 1.09 | 1.21 | 1.11 | |
562 | 6.8 | 18 | Bestrophin-3 = BEST3 | 38 | 1.26 | 1.31 | 1.42 | 1.04 | 1.13 | 1.09 | |
563 | 6.0 | 18 | Actin, cytoplasmic 2 = ACTG2 | 137 | −1.53 | −1.74 | 1.44 | −1.14 | 2.20 | 2.50 | |
567 | 6.9 | 18 | Glutathione S-transferase P = GSTP1 | 286 | −1.02 | −1.09 | 1.22 | −1.07 | 1.24 | 1.33 | |
568 | 3.6 | 18 | Transcriptional repressor protein YY1 = YY1 | 39 | 1.47 | 1.16 | 1.13 | −1.27 | −1.30 | −1.03 | |
572 | 5.8 | 18 | Ferritin light chain = FTL | 74 | −1.01 | −1.06 | −1.26 | −1.04 | −1.25 | −1.20 | |
574 | 4.7 | 17 | Tubulin beta-1 = TUBB1 | 39 | 1.21 | −1.04 | 1.18 | −1.26 | −1.03 | 1.22 | |
582 | 4.0 | 17 | Keratin, type II cytoskeletal 1 = KRT1 | 116 | 1.34 | −1.14 | −1.20 | −1.53 | −1.60 | −1.05 | |
583 | 4.1 | 17 | Isoform 3 of mitoch carrier homolog 1 = MTCH1 | 39 | −1.00 | 1.09 | −1.07 | 1.09 | −1.07 | −1.16 | |
588 | 3.9 | 17 | Alpha-actinin-1 = ACTN1 | 222 | 1.44 | −1.06 | −1.04 | −1.53 | −1.50 | 1.02 | |
591 | 7.6 | 17 | Heat shock 70 kDa protein 1A/1B = HSPA1A | 43 | 1.01 | 1.26 | 1.42 | 1.25 | 1.41 | 1.13 | |
592 | 4.4 | 17 | Myosin regulatory light chain 12B = MYL12B | 320 | 1.43 | 1.28 | 1.38 | −1.12 | −1.04 | 1.08 | |
593 | 4.5 | 17 | Actin, cytoplasmic 1 = ACTB | 65 | 1.63 | 1.11 | 1.24 | −1.46 | −1.32 | 1.11 | |
595 | 8.3 | 17 | Protein S100-A8 = S100A8 | 60 | 1.59 | −1.07 | 1.12 | −1.70 | −1.43 | 1.19 | |
603 | 4.8 | 16 | Annexin = ANXA5 | 111 | −1.20 | 1.19 | 1.02 | 1.44 | 1.23 | −1.17 | |
605 | 5.6 | 16 | ATP synthase subunit alpha = ATP5F1A | 95 | −1.03 | −1.24 | −1.08 | −1.20 | −1.05 | 1.14 | |
607 | 6.7 | 16 | Actin-related protein 2/3 complex subunit 3 = ARPC3 | 50 | 1.06 | 1.21 | 1.09 | 1.14 | 1.02 | −1.12 | |
610 | 4.5 | 16 | Actin, cytoplasmic 2 = ACTG1 | 103 | 1.32 | −1.01 | 1.08 | −1.34 | −1.22 | 1.10 | |
611 | 7.1 | 16 | Cofilin 1 (Non-muscle), isoform CRA_a = CFL1 | 61 | 1.15 | 1.12 | 1.07 | −1.03 | −1.07 | −1.04 | |
612 | 4.2 | 16 | Myosin regulatory light polypeptide 9 = MYL9 | 233 | 1.19 | 1.26 | 1.10 | 1.06 | −1.08 | −1.15 | |
613 | 4.6 | 16 | Actin, cytoplasmic 2 = ACTG1 | 61 | 1.45 | 1.04 | 1.13 | −1.39 | −1.29 | 1.08 | |
614 | 8.7 | 16 | Rho GDP-dissociation inhibitor 2 = ARHGDIB | 130 | 1.01 | 1.09 | 1.13 | 1.08 | 1.11 | 1.03 | |
620 | 4.3 | 16 | Isoform 3 of mitoch carrier homolog 1 = MTCH1 | 38 | 1.33 | 1.00 | 1.22 | −1.33 | −1.09 | 1.21 | |
621 | 4.6 | 16 | Actin, cytoplasmic 1 = ACTB | 153 | 1.20 | 1.02 | −1.04 | −1.18 | −1.25 | −1.06 | |
623 | 5.2 | 16 | Actin, cytoplasmic 2 = ACTG1 | 130 | 1.30 | 1.02 | 1.15 | −1.27 | −1.14 | 1.12 | |
627 | 5.5 | 15 | Annexin A6 = ANXA6 | 103 | −1.25 | 1.08 | −1.27 | 1.35 | −1.02 | −1.38 | |
628 | 3.8 | 15 | Myosin light polypeptide 6 = MYL6 | 302 | 1.30 | 1.08 | 1.01 | −1.20 | −1.29 | −1.07 | |
629 | 4.4 | 15 | Actin, cytoplasmic 1 = ACTB | 65 | 1.24 | −1.07 | −1.30 | −1.32 | −1.61 | −1.22 | |
630 | 8.1 | 15 | Peptidyl-prolyl cis-trans isomerase A = PPIA | 199 | −1.04 | −1.03 | 1.02 | 1.01 | 1.06 | 1.05 | |
632 | 9.0 | 15 | Peptidyl-prolyl cis-trans isomerase A = PPIA | 235 | −1.01 | −1.04 | −1.09 | −1.03 | −1.08 | −1.05 | |
637 | 4.4 | 15 | Heterogeneous nuclear ribonucleoprotein K = HNRNPK | 42 | 1.38 | 1.10 | −1.23 | −1.25 | −1.70 | −1.36 | |
640 | 5.1 | 15 | Actin, cytoplasmic 1 = ACTB | 193 | 1.45 | 1.21 | 1.05 | −1.20 | −1.39 | −1.16 | |
642 | 4.2 | 15 | Actin, cytoplasmic 1 = ACTB | 139 | 1.05 | 1.06 | 1.09 | 1.01 | 1.04 | 1.03 | |
644 | 7.7 | 15 | Keratin, type I cytoskeletal 10 = KRT10 | 47 | 1.16 | 1.18 | −1.01 | 1.01 | −1.17 | −1.19 | |
646 | 4.0 | 15 | Myosin light polypeptide 6 = MYL6 | 369 | 1.16 | 1.05 | 1.01 | −1.11 | −1.15 | −1.04 | |
648 | 8.4 | 15 | Bestrophin-3 = BEST3 | 160 | 1.17 | 1.21 | 1.13 | 1.03 | −1.04 | −1.07 | |
650 | 7.3 | 15 | Heterogeneous nuclear ribonucleoprotein A1 = HNRNPA1 | 102 | −1.04 | −1.15 | 1.36 | −1.11 | 1.41 | 1.56 | |
657 | 4.8 | 15 | Actin, cytoplasmic 1 = ACTB | 108 | 1.25 | −1.01 | −1.31 | −1.26 | −1.63 | −1.29 | |
659 | 4.5 | 14 | Actin, cytoplasmic 2 = ACTG1 | 255 | 1.37 | −1.06 | −1.01 | −1.45 | −1.39 | 1.04 | |
662 | 4.7 | 14 | Actin, cytoplasmic 1 = ACTB | 263 | 1.55 | 1.08 | −1.01 | −1.43 | −1.56 | −1.10 | |
664 | 7.0 | 14 | Actin, cytoplasmic 2 = ACTG1 | 41 | 1.53 | 1.21 | −1.02 | −1.26 | −1.55 | −1.23 | |
665 | 4.5 | 14 | Actin, cytoplasmic 1 = ACTB | 233 | 1.56 | 1.05 | 1.12 | −1.49 | −1.40 | 1.06 | |
666 | 3.8 | 14 | Isoform 3 of mitoch carrier homolog 1 = MTCH1 | 32 | 1.34 | 1.07 | 1.12 | −1.25 | −1.19 | 1.05 | |
671 | 8.7 | 14 | Bestrophin-3 = BEST3 | 40 | 1.21 | 1.02 | −1.09 | −1.19 | −1.32 | −1.11 | |
673 | 4.0 | 14 | Isoform H14 of Myeloperoxidase = MPO | 288 | −1.16 | 1.07 | 1.09 | 1.24 | 1.26 | 1.02 | |
675 | 4.3 | 13 | Actin, cytoplasmic 1 = ACTB | 232 | −1.05 | 1.04 | 1.10 | 1.09 | 1.15 | 1.05 | |
676 | 4.4 | 13 | Actin, cytoplasmic 1 = ACTB | 225 | 1.06 | −1.00 | −1.01 | −1.07 | −1.07 | −1.00 | |
677 | 4.6 | 13 | Actin, cytoplasmic 1 = ACTB | 244 | −1.01 | −1.01 | −1.17 | 1.00 | −1.15 | −1.16 | |
679 | 7.7 | 13 | Actin, cytoplasmic 1 = ACTB | 103 | 1.09 | −1.31 | −1.32 | −1.43 | −1.44 | −1.01 | |
680 | 7.2 | 13 | Myosin light polypeptide 6 = MYL6 | 421 | −1.11 | −1.04 | −1.14 | 1.06 | −1.03 | −1.09 | |
683 | 5.0 | 13 | Histone H4 = HIST1H4A | 242 | 1.01 | −1.37 | −1.43 | −1.39 | −1.45 | −1.04 | |
685 | 7.6 | 13 | Hemoglobin subunit beta = HBB | 255 | −1.14 | −1.55 | −1.56 | −1.35 | −1.36 | −1.00 | |
686 | 4.8 | 13 | Centromere protein H = CENPH | 38 | −1.19 | −1.01 | 1.01 | 1.18 | 1.21 | 1.02 | |
690 | 7.9 | 13 | Hemoglobin subunit beta = HBB | 384 | −1.15 | −1.43 | −1.51 | −1.24 | −1.31 | −1.06 | |
696 | 3.8 | 13 | Histone H4 = HIST1H4A | 60 | −1.31 | 1.04 | −1.11 | 1.36 | 1.18 | −1.16 | |
697 | 7.8 | 12 | Bestrophin-3 = BEST3 | 39 | 1.16 | −1.49 | −1.53 | −1.73 | −1.77 | −1.03 | |
698 | 4.6 | 12 | Enolase = ENO3 | 63 | −1.24 | −1.15 | −1.01 | 1.07 | 1.22 | 1.14 | |
703 | 7.7 | 12 | LVV-hemorphin-7 = HBB | 41 | −1.13 | −1.85 | −1.29 | −1.64 | −1.14 | 1.44 | |
704 | 8.1 | 12 | SH3 domain-binding glutamic acid-rich-like protein 2 = SH3BGRL2 | 314 | 1.24 | −1.60 | −1.31 | −1.99 | −1.62 | 1.23 | |
706 | 6.7 | 12 | Protein S100-A11 = S100A11 | 260 | −1.39 | −1.12 | −1.10 | 1.25 | 1.27 | 1.02 | |
707 | 4.6 | 12 | Isoform 3 of mitoch carrier homolog 1 = MTCH1 | 41 | 1.17 | −1.20 | −1.12 | −1.40 | −1.31 | 1.07 | |
711 | 4.1 | 12 | Nostrin = NOSTRIN | 44 | −1.49 | −1.07 | −1.01 | 1.38 | 1.47 | 1.07 | |
712 | 5.1 | 12 | Myotrophin = MTPN | 146 | −1.29 | −1.16 | −1.66 | 1.11 | −1.29 | −1.44 | |
713 | 4.9 | 12 | Ras-related protein Ral-B = RALB | 37 | 1.12 | −1.15 | −1.17 | −1.29 | −1.31 | −1.02 | |
718 | 4.5 | 11 | Ras-related protein Ral-B = RALB | 155 | 1.20 | −1.37 | −1.14 | −1.65 | −1.37 | 1.20 | |
721 | 3.9 | 11 | Protein S100-A6 = S100A6 | 68 | −1.31 | 1.00 | −1.11 | 1.31 | 1.18 | −1.11 | |
723 | 8.7 | 11 | Transcriptional repressor protein YY1 = YY1 | 41 | 1.35 | −1.39 | −1.15 | −1.87 | −1.55 | 1.21 | |
725 | 7.7 | 11 | Histone H4 = HIST1H4A | 70 | 1.08 | −1.30 | −1.29 | −1.40 | −1.39 | 1.01 | |
732 | 4.7 | 11 | Thrombospondin-1 = THBS1 | 115 | 1.61 | −1.19 | −1.31 | −1.91 | −2.11 | −1.10 | |
735 | 4.3 | 10 | SH3 domain-binding glutamic acid-rich-like protein 3 = SH3BGRL3 | 349 | −1.06 | 1.11 | −1.18 | 1.18 | −1.11 | −1.31 | |
737 | 4.0 | 10 | ATP synthase subunit alpha, mitoch = ATP5A1 | 173 | −1.16 | −1.11 | −1.06 | 1.04 | 1.10 | 1.05 | |
738 | 3.9 | 10 | Keratin, type II cytoskeletal 1 = KRT1 | 96 | 1.07 | −1.10 | −1.09 | −1.17 | −1.16 | 1.01 | |
740 | 4.9 | 0 | Urea transporter 1 = SLC14A1 | 39 | 1.33 | −1.19 | −1.02 | −1.59 | −1.36 | 1.17 | |
744 | 4.4 | 0 | Ras-related protein Rap-1b = RAP1B | 58 | −1.02 | 1.09 | −1.11 | 1.11 | −1.09 | −1.21 | |
745 | 4.2 | 0 | SH3 domain-binding glutamic acid-rich-like protein 3 = SH3BGRL3 | 171 | −1.07 | −1.08 | −1.08 | −1.02 | −1.02 | 1.00 | |
756 | 5.3 | 45 | Actin, cytoplasmic 1 = ACTB | 343 | −1.32 | −1.07 | −1.14 | 1.23 | 1.16 | −1.06 | |
758 | 5.4 | 38 | Actin, cytoplasmic 1 = ACTB | 624 | −1.30 | −1.25 | −1.10 | 1.04 | 1.19 | 1.14 | |
759 | 4.8 | 15 | Actin, cytoplasmic 2 = ACTG1 | 263 | 1.27 | 1.05 | −1.02 | −1.20 | −1.29 | −1.07 | |
761 | 4.8 | 16 | Keratin, type II cytoskeletal 1 = KRT1 | 789 | 1.24 | −1.03 | −1.05 | −1.27 | −1.30 | −1.02 | |
762 | 4.9 | 16 | Actin, cytoplasmic 2 = ACTG1 | 221 | 1.34 | −1.08 | 1.05 | −1.44 | −1.28 | 1.13 | |
763 | 5.7 | 20 | Actin, cytoplasmic 1 = ACTB | 416 | −1.06 | −1.12 | −1.10 | −1.06 | −1.04 | 1.02 | |
769 | 5.4 | 14 | Actin, cytoplasmic 1 = ACTB | 44 | 1.07 | −1.06 | −1.65 | −1.13 | −1.76 | −1.56 | |
772 | 5.4 | 13 | Histone H4 = HIST1H4A | 193 | −1.36 | −1.07 | −1.18 | 1.27 | 1.15 | −1.11 | |
774 | 3.7 | 16 | Calmodulin = CALM1 | 367 | −1.11 | 1.13 | 1.10 | 1.25 | 1.22 | −1.02 | |
779 | 4.6 | 28 | Actin, cytoplasmic 1 = ACTB | 428 | 1.16 | −1.01 | 1.02 | −1.16 | −1.14 | 1.02 | |
781 | 4.6 | 27 | Actin, cytoplasmic 1 = ACTB | 342 | 1.04 | −1.11 | −1.09 | −1.16 | −1.14 | 1.01 | |
782 | 4.7 | 27 | Tropomyosin alpha-4 = TPM4 | 665 | 1.48 | −1.04 | 1.14 | −1.54 | −1.31 | 1.18 | |
785 | 9.0 | 13 | Profilin-1 = PFN1 | 157 | 1.06 | −1.13 | −1.02 | −1.19 | −1.08 | 1.10 | |
787 | 5.3 | 12 | Ras-related protein Ral-B = RALB | 35 | −1.10 | −1.01 | −1.37 | 1.09 | −1.24 | −1.36 | |
796 | 5.3 | 13 | Keratin, type II cytoskeletal 1 = KRT1 | 127 | 1.29 | −1.07 | −1.54 | −1.38 | −1.99 | −1.43 | |
797 | 5.3 | 13 | Keratin, type II cuticular Hb3 = KRT83 | 165 | 1.08 | 1.08 | −1.59 | 1.00 | −1.72 | −1.72 | |
804 | 7.0 | 10 | LVV-hemorphin-7 = HBB | 122 | 1.26 | −1.12 | −1.28 | −1.41 | −1.61 | −1.15 | |
808 | 8.1 | 11 | Platelet basic protein = PPBP | 90 | 1.43 | −1.33 | −1.20 | −1.90 | −1.71 | 1.11 | |
809 | 8.2 | 11 | Protein S100-A8 = S100A8 | 66 | 1.23 | −1.13 | −1.06 | −1.39 | −1.30 | 1.07 | |
811 | 8.9 | 11 | Platelet basic protein = PPBP | 216 | 1.25 | 1.02 | 1.05 | −1.23 | −1.19 | 1.03 | |
816 | 7.5 | 78 | Actin, cytoplasmic 1 = ACTB | 454 | −1.22 | 1.05 | −1.34 | 1.28 | −1.10 | −1.41 | |
825 | 8.7 | 72 | Isoform Delta of Lactotransferrin = LTF | 147 | −1.44 | −1.07 | −1.04 | 1.34 | 1.38 | 1.02 | |
845 | 4.1 | 68 | Isoform 4 of Regulating synaptic membrane exocytosis protein 2 = RIMS2 | 37 | 1.24 | −1.29 | −1.03 | −1.61 | −1.29 | 1.25 | |
854 | 8.7 | 67 | Kaliocin-1 = LTF | 46 | −1.09 | −1.12 | −1.15 | −1.02 | −1.06 | −1.03 | |
863 | 5.5 | 13 | Keratin, type I cytoskeletal 9 = KRT9 | 475 | −1.79 | −1.38 | −1.44 | 1.30 | 1.25 | −1.04 | |
866 | 4.9 | 10 | Protein S100-A6 = S100A6 | 39 | 1.46 | −1.16 | −1.15 | −1.70 | −1.68 | 1.01 | |
867 | 4.9 | 0 | LVV-hemorphin-7 = HBB | 65 | 1.26 | −1.21 | −1.19 | −1.52 | −1.49 | 1.02 | |
868 | 3.6 | 16 | Calmodulin = CALM1 | 343 | −1.01 | 1.06 | 1.03 | 1.08 | 1.05 | −1.03 | |
870 | 3.6 | 16 | Calmodulin = CALM1 | 63 | 1.45 | 1.11 | 1.09 | −1.30 | −1.32 | −1.02 | |
871 | 3.7 | 16 | Calmodulin = CALM1 | 195 | 1.33 | 1.11 | −1.04 | −1.20 | −1.39 | −1.15 | |
877 | 3.8 | 55 | Stromal interaction molecule 2 = STIM2 | 39 | 1.31 | −1.08 | −1.09 | −1.42 | −1.43 | −1.00 | |
878 | 6.9 | 10 | Protein S100-A11 | 246 | −1.13 | −1.35 | −1.11 | −1.20 | 1.01 | 1.22 | |
879 | 6.9 | 11 | LVV-hemorphin-7 = HBB | 213 | −1.02 | −1.22 | −1.27 | −1.20 | −1.25 | −1.04 | |
884 | 4.4 | 22 | Tropomyosin 3 = TPM3 | 421 | 1.48 | −1.07 | 1.03 | −1.58 | −1.43 | 1.10 | |
885 | 4.4 | 21 | Tropomyosin 3 = TPM3 | 62 | 1.20 | 1.06 | 1.04 | −1.13 | −1.14 | −1.02 | |
889 | 7.8 | 70 | Isoform 2 of Fibrinogen alpha = FGA | 165 | −1.04 | 1.07 | −1.29 | 1.12 | −1.24 | −1.38 | |
890 | 4.9 | 16 | Actin, alpha 1, skeletal muscle = ACTA1 | 124 | 1.24 | 1.02 | 1.02 | −1.22 | −1.21 | 1.01 | |
891 | 5.0 | 15 | Actin, cytoplasmic 2 = ACTG1 | 113 | 1.23 | 1.05 | 1.02 | −1.16 | −1.20 | −1.03 | |
892 | 7.6 | 13 | Annexin A2 = ANXA2 | 109 | −1.32 | −1.37 | −1.11 | −1.04 | 1.19 | 1.23 | |
896 | 4.3 | 14 | Actin, cytoplasmic 1 = ACTB | 198 | 1.15 | −1.14 | −1.06 | −1.31 | −1.22 | 1.07 | |
897 | 4.3 | 14 | Actin, cytoplasmic 1 = ACTB | 242 | 1.15 | 1.02 | 1.10 | −1.13 | −1.05 | 1.08 | |
901 | 8.6 | 13 | Hemoglobin subunit beta = HBB | 254 | 1.17 | −1.26 | −1.22 | −1.47 | −1.42 | 1.04 | |
902 | 8.6 | 13 | Hemoglobin subunit beta = HBB | 350 | 1.02 | −1.28 | −1.19 | −1.30 | −1.21 | 1.07 | |
903 | 8.6 | 13 | Hemoglobin subunit beta = HBB | 353 | 1.18 | −1.26 | −1.11 | −1.49 | −1.31 | 1.14 | |
904 | 8.3 | 13 | Hemoglobin subunit beta = HBB | 349 | 1.07 | −1.29 | −1.37 | −1.37 | −1.46 | −1.07 | |
905 | 8.3 | 13 | Hemoglobin subunit beta = HBB | 330 | −1.09 | −1.32 | −1.30 | −1.21 | −1.19 | 1.02 | |
907 | 5.6 | 38 | Actin, cytoplasmic 1 7 = ACTB | 203 | −1.34 | −1.22 | −1.06 | 1.10 | 1.26 | 1.14 | |
911 | 4.7 | 10 | SH3 domain-binding glutamic acid-rich-like protein 3 = SH3BGRL3 | 354 | 1.39 | −1.02 | −1.23 | −1.42 | −1.72 | −1.21 | |
913 | 4.7 | 117 | Rho guanine nucleotide exchange factor 25 = ARHGEF25 | 32 | 1.09 | −1.20 | −1.07 | −1.30 | −1.16 | 1.12 | |
914 | 5.1 | 85 | Actin, cytoplasmic 2 = ACTG1 | 157 | 1.15 | −1.08 | 1.02 | −1.24 | −1.12 | 1.10 | |
KEY | Protein Score ≥ 62 | 7.00 | 1.00 | 1.00 | 10.00 | 6.00 | 4.00 | ||||
5.00 | 5.00 | 7.00 | 20.00 | 22.00 | 4.00 |
PBMC SNO signature in Chagas disease.
We utilized a new set of PBMC from NH, CA, and CS cohorts (
Biotin switch assay for verification of abundance and SNO levels of ACTG and FLNA in Chagas disease.
We first examined the changes in abundance of ACTG and FLNA in ChD patients and NH controls by an ELISA. In ChD CA patients (vs. NH controls), we observed 79% (
For the detection of SNO modification levels of proteins in PBMC lysates, the reduced thiol groups were blocked, and then SNO-modified proteins were reduced to make these available for binding with biotin, and detection by avidin-conjugated horseradish peroxidase method. These data showed >80% changes in SNO levels of ACTG in PBMC from ChD CA and ChD CS patients (vs. NH subjects, Figure
The differences in the observations between ELISA/Biotin Switch Assay and BD-fluor/2D-GE based proteomic study can at least partially be explanined by the fact that 2D-GE/proteomic approach identifies the full length protein as well as derived peptides as individual spots that may change in concentration depending on their stability and/or degradation, while ELISA did not discriminate between the full length and smaller peptides of these proteins; the level of detection will depend on the epitopes that the coated antibody recognizes. Secondly, Biotin switch assay simply detects the SNO-modified proportion of a protein but does not take into consideration the changes in its abundance, while RoR approach normalizes SNO level against the protein concentration in experimental sample and then also derives the values in comparison to control data. At present no other reliable and easy to use assays exist that can detect the SNO modification level per unit protein. Despite the discrepancies between the two approaches, our results in Figure
For this analysis, we log2 transformed the protein spot intensities to ensure that biologically relevant proportional changes are captured between the different groups (Feng et al.,
Comparing Asc−/Asc+ values of ChD CA vs. NH groups, we identified nine protein spots that exhibited differential SNO modification level (
MARS modeling of SNO-modified proteins for classification of clinically asymptomatic ChD patients. For this, log2 transformed protein spot intensities on each of the Asc+ and Asc− gels of PBMC lysates from ChD clinically asymptomatic (ChD CA,
Comparing Asc−/Asc+ values of ChD CS vs. NH groups, we identified 11 protein spots that exhibited significant differences in SNO modification level (
MARS modeling of SNO-modified proteins for classification of clinically symptomatic ChD patients. As in Figure
Together, the results presented in Figures
We employed Ingenuity Pathway Analysis software to determine molecular and biological functions, as well as the important pathways and networks involved in Chagas disease development. Input to IPA were the RoR values (
SNO profile of cell death network in Chagas disease (ChD). PBMC SNO proteome of ChD clinically asymptomatic (ChD CA,
IPA analysis of RoR proteome datasets also predicted a putative differential SNO profile of proteins involved in recruitment of immune cells, i.e., leukocytes, neutrophils, and phagocytes, and overall development of inflammatory response in ChD CA patients (13 molecules,
SNO profile of inflammation and immune responses network in Chagas disease. As in Figure
In this discovery proteomic study, we have focused on identifying the S-nitrosylation profile of PBMC from seropositive ChD patients with or without clinical disease in comparison with seronegative healthy subjects (
We noted increased SNO modification (–ve RoR value) of three proteins (THBS1, S100A6, abd SH3BGRL2) in ChD patients in this study, as well in congestive heart failure (CHF) patients of idiopathic etiology in a previous study (Koo et al.,
We performed MARS analysis of the datasets to understand the diagnostic potential of the SNO proteome datasets in identifying the disease status in Chagas patients (Figures
Ingenuity Pathway Analysis of the proteome datasets (RoR values, fold change |≥1.2|,
In summary, we have presented unbiased SNO proteomic analysis of PBMC of Chagas disease patients in this study. We have identified the possible pathologic mechanisms in disease progression that involve immune cell activation and cell death. MARS-modeling identified a panel of protein spots that if monitored in infected individuals, would have high degree of success in predicting risk of clinical disease development.
MZ collected and processed the human samples. MZ, JW, HS, S-JK, and NG performed experiments, analyzed data, and wrote manuscript. NB, AN, JN, VB, FI and RL recruited patients, performed physical/clinical exam, serology, and other examination, and clinically characterized patients, everyone was involved in study design, and reviewing the manuscript. NG provided financial support for carrying out the experiments. RL and NB supported the patient exam.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are thankful for the support provided in patients' sample collection and serology tests by Dr. Ines Vidal and lab technicians of the Central Laboratory at the San Bernardo Hospital and the Papa Francisco Hospital in Salta, Argentina.