Quantitative Proteomic Approach Targeted to Fibrinogen β Chain in Tissue Gastric Carcinoma
Abstract
:1. Introduction
2. Results
2.1. Patients and Disease Characteristics
2.2. One-Dimension Immunoblotting
2.3. Validation of Fibrinogen β Chain (FGB) by Mass Spectrometry
2.4. Cross-Reaction of FGB on Two-Dimensional Electrophoresis (2DE) Maps of Gastric Tissue
2.5. Differential Expression of FGB in Gastric Cancer Tissues
2.6. Correlation of Blood Count Parameters and FGB Abundance
3. Discussion
4. Materials and Methods
4.1. Patients and Tissues
4.2. Blood Sample Analysis
4.3. Sample Preparation and Grouping
4.4. One-Dimensional Electrophoresis (1DE) and Immunoblotting Anti-Fibrinogen β Chain
4.5. Validation of the 1DE Bands Cross-Reacting with Fibrinogen β Chain
4.6. Two-Dimensional Difference in Gel Electrophoresis (2D-DIGE) and Immunoblotting Anti-Fibrinogen β Chain
4.7. Database Searches for Fibrinogen β Chain Sequence and Post-Translational Modifications
4.8. Image Analysis and Statistics
4.9. Correlation Analysis of Blood Parameters
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
1DE | one-dimensional electrophoresis |
2DE | two-dimensional electrophoresis |
ALC | absolute lymphocyte count |
ANC | absolute neutrophil count |
APTT | activated partial thromboplastin time |
APTT RATIO | ratio of APPT |
BVA | biological variation analysis |
DIA | decyder differential in-gel analysis |
DIGE | difference gel electrophoresis |
FGB | fibrinogen β chain |
FIB | fibrinogen level |
GC | gastric cancer |
IEF | isoelectrofocusing |
N/R | ANC/ALC ratio |
PLT | platelet count |
PT | prothrombin time |
WBC | white blood cells |
Appendix A
Patient nr. | Age | Male/Female a | uT | uN b | Histological Classification b | Anatomical Subsites | Tissue c | Cye Dye | Gel nr. |
---|---|---|---|---|---|---|---|---|---|
1 | 54 | M | 3 | N+ | diffuse | corpus | N | cy3 | 1 |
T | cy5 | ||||||||
2 | 50 | F | 3 | N+ | diffuse | antrum | N | cy3 | 2 |
T | cy5 | ||||||||
3 | 44 | F | 3 | N+ | diffuse | corpus | N | cy3 | 3 |
T | cy5 | ||||||||
4 | 39 | F | 3 | N+ | diffuse | corpus | N | cy3 | 4 |
T | cy5 | ||||||||
5 | 68 | M | 3 | N0 | diffuse | corpus | N | cy3 | 5 |
6 | 52 | M | 3 | N+ | n.a. | corpus | T | cy5 | |
7 | 55 | M | 3 | N+ | diffuse | corpus | N | cy5 | 6 |
T | cy3 | ||||||||
8 | 81 | F | 3 | N+ | intestinal | antrum | N | cy5 | 7 |
T | cy3 | ||||||||
9 | 74 | M | 3 | N+ | diffuse | antrum | N | cy5 | 8 |
T | cy3 | ||||||||
10 | 65 | M | 4 | N+ | diffuse | corpus | N | cy5 | 9 |
T | cy3 | ||||||||
11 | 71 | F | 3 | N0 | diffuse | antrum | N | cy5 | 10 |
12 | 69 | M | 3 | N+ | intestinal | corpus | T | cy3 | |
13 | 60 | F | 4 | n.a. | other | corpus | N | cy5 | 11 |
T | cy3 | ||||||||
14 | 68 | F | 4 | N0 | intestinal | corpus | N | cy5 | 12 |
T | cy3 | ||||||||
15 | 54 | M | 3 | N+ | diffuse | cardia | N | cy5 | 13 |
T | cy3 | ||||||||
16 | 70 | F | 2 | n.a. | intestinal | antrum | N | cy5 | 14 |
T | cy3 | ||||||||
17 | 76 | F | 1 | N0 | intestinal | antrum | N | cy3 | 15 |
T | cy5 | ||||||||
18 | 59 | M | 1 | N0 | other | cardia | N | cy3 | 16 |
T | cy5 | ||||||||
19 | 66 | F | 1 | N0 | diffuse | corpus | N | cy5 | 17 |
T | cy3 | ||||||||
20 | 48 | M | 2 | N0 | intestinal | corpus | N | cy5 | 18 |
T | cy3 | ||||||||
21 | 36 | F | 1 | N0 | intestinal | corpus | N | cy5 | 19 |
T | cy3 | ||||||||
22 | 59 | M | 1 | N+ | diffuse | corpus | N | cy5 | 19 |
T | cy3 | ||||||||
23 | 69 | F | 3 | N0 | n.a. | corpus | N | cy3 | 20 |
T | cy5 | ||||||||
24 | 58 | M | 3 | N+ | diffuse | corpus | N | cy3 | 21 |
T | cy5 | ||||||||
25 | 45 | F | 3 | N+ | diffuse | corpus | N | cy5 | 22 |
T | cy3 | ||||||||
26 | 68 | M | 1 | N0 | diffuse | antrum | N | cy5 | 23 |
T | cy3 | ||||||||
27 | 50 | F | 1 | N0 | diffuse | antrum | N | cy5 | 24 |
T | cy3 | ||||||||
28 | 55 | F | 3 | N+ | diffuse | corpus | N | cy5 | 25 |
T | cy3 |
Patient nr. a | WBC × 109/L | PLT × 109/L | ANC × 109/L | ALC × 109/L | N/R | PT-INR | FIB g/L b | APTTs | APTT RATIO |
---|---|---|---|---|---|---|---|---|---|
1 | 4.65 | 221 | 3 | 1.3 | 2.31 | 0.93 | 2.91 | 27 | 0.83 |
2 | 1.19 | 57 | 0.7 | 0.4 | 1.75 | 0.93 | 2.76 | 26 | 0.86 |
3 * | 5.23 | 379 | 4.5 | 0.7 | 6.43 | 1.13 | 2.76 | 26 | 0.88 |
4 | 7.9 | 272 | 4.2 | 2.6 | 1.62 | 1 | 3.54 | 33 | 1.1 |
5 | 7.61 | 174 | 4.1 | 2.9 | 1.41 | 0.99 | 2.20 | 28 | 0.93 |
6 * | 8.95 | 624 | 5.3 | 2.7 | 1.96 | 0.95 | n.a. | 27 | 0.89 |
7 | 6.29 | 182 | 3.4 | 2 | 1.70 | 0.99 | 2.95 | 34 | 1.14 |
8 | 4.74 | 184 | 2.6 | 1.8 | 1.44 | 1 | 2.76 | 31 | 1.03 |
9 * | 7.19 | 283 | 4.3 | 2 | 2.15 | 0.96 | 3.34 | 29 | 0.95 |
10 | 9.61 | 137 | 7.8 | 1.1 | 7.09 | 1.03 | 3.01 | 24 | 0.79 |
11 | 7.92 | 330 | 5.3 | 2 | 2.65 | 0.99 | n.a. | 32 | 1.04 |
12 * | 7.57 | 421 | 3.9 | 3.1 | 1.26 | 0.95 | 2.96 | 31 | 1.03 |
13 | 8.12 | 231 | 6.1 | 1.2 | 5.08 | 1.02 | 3.37 | 25 | 0.83 |
14 | 5.11 | 245 | 2.7 | 1.9 | 1.42 | 0.92 | 3.57 | 24 | 0.79 |
15 | 6.74 | 212 | 5.1 | 1.1 | 4.64 | 1.04 | 3.68 | 32 | 1.06 |
16 | 4.8 | 234 | 2.4 | 2 | 1.20 | 1.06 | 3.03 | 29 | 0.95 |
17 | 4,1 | 215 | 2.4 | 1.3 | 1.85 | 1.03 | 3.45 | 30 | 1.01 |
18 * | 7.72 | 283 | 4.1 | 2.8 | 1.46 | 1.03 | 3.37 | 32 | 1.07 |
19 | 7.63 | 233 | 5.5 | 1.6 | 3.44 | 0.97 | 2.16 | 27 | 0.89 |
20 * | 6.71 | 345 | 3.3 | 2.7 | 1.22 | 1.02 | 2.56 | 29 | 0.97 |
21 | 5.05 | 235 | 3.2 | 1.6 | 2.00 | 0.95 | 2.56 | 27 | 0.91 |
22 | 4.51 | 177 | 2.6 | 1.5 | 1.73 | 1.01 | 2.26 | 28 | 0.95 |
Gel Portion nr.a | Database | Accession | Description | Score/Seq. Coverage % | Start-End | Peptide | Matches/Sequences |
---|---|---|---|---|---|---|---|
4 | NCBInr | gi|119625336 | Fibrinogen β chain, isoform CRA_b | 239/29 | 42–48 50–69 161–175 197–203 220–226 228–244 | R.GHRPLDK.K K.REEAPSLRPAPPPISGGGYR.A K.DNENVVNEYSSELEK.H R.SILENLR.S K.ECEEIIR.K K.GGETSEMYLIQPDSSVK.D | 11/7 |
5 | SwissProt | FIBB_HUMAN | Fibrinogen β chain | 376/28 | 45–51 53–72 164–178 200–206 212–224 240–246 248–267 301–313 354–367 368–374 411–421 484–491 | R.GHRPLDK.K K.REEAPSLRPAPPPISGGGYR.A K.DNENVVNEYSSELEK.H R.SILENLR.S K.LESDVSAQMEYCR.T K.ECEEIIR.K K.GGETSEMYLIQPDSSVKPYR.V K.QGFGNVATNTDGK.N K.AHYGGFTVQNEANK.Y K.YQISVNK.Y R.DNDGWLTSDPR.K K.IRPFFPQQ.- | 19/12 |
7 * | SwissProt | FIBB_HUMAN | Fibrinogen β chain | 269/15 | 164–178 212–224 225–239 301–313 354–367 484–491 | K.DNENVVNEYSSELEK.H K.LESDVSAQMEYCR.T R.TPCTVSCNIPVVSGK.E K.QGFGNVATNTDGK.N K.AHYGGFTVQNEANK.Y K.IRPFFPQQ.- | 6/5 |
8 * | SwissProt | FIBB_HUMAN | Fibrinogen β chain | 340/19 | 164–178 212–224 225–239 240–246 248–267 301–313 354–367 | K.DNENVVNEYSSELEK.H K.LESDVSAQMEYCR.T R.TPCTVSCNIPVVSGK.E K.ECEEIIR.K K.GGETSEMYLIQPDSSVKPYR.V K.QGFGNVATNTDGK.N K.AHYGGFTVQNEANK.Y | 8/7 |
9 * | SwissProt | FIBB_HUMAN | Fibrinogen β chain | 477/21 | 164–178 212–224 225–239 240–246 248–267 301–313 354–367 484–491 | K.DNENVVNEYSSELEK.H K.LESDVSAQMEYCR.T R.TPCTVSCNIPVVSGK.E K.ECEEIIR.K K.GGETSEMYLIQPDSSVKPYR.V K.QGFGNVATNTDGK.N K.AHYGGFTVQNEANK.Y K.IRPFFPQQ.- | 13/11 |
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Characteristic | uT1–T2 | uT3–T4 | p-Value |
---|---|---|---|
Gender | n.s. | ||
Male | 4 | 9 | |
Female | 5 | 10 | |
Age, years | n.s. | ||
<50 | 3 | 4 | |
>50 | 6 | 15 | |
Histological type | n.s. | ||
Intestinal | 4 | 4 | |
Diffuse | 4 | 13 | |
Other | 1 | 2 | |
Location | n.s. | ||
Corpus | 4 | 14 | |
Antrum | 4 | 4 | |
Cardias | 1 | 1 |
Parameter | Mean (±SD) |
---|---|
WBC (×109/L) | 6.33 ± 1.96 |
ANC(×109/L) | 3.93 ± 1.54 |
ALC (×109/L) | 1.83 ± 0.74 |
N/R | 2.54 ± 1.71 |
PLT (×109/L) | 258 ± 114.90 |
PT-INR | 1.00 ± 0.05 |
FIB g/L | 2.97 ± 46 |
APTTs | 29 ± 3 |
Comparison Groups | Spots | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
383 | 397 | 398 | 400 | 393 | 396 | 402 | 403 | 404 | 405 | 501 | 526 | 548 | ||
• | N “Group II” versus N “Group I” | 1 | −1.6 | −1.3 | −1.0 | 1.1 | 1.1 | 1.5 | 1.2 | −1.0 | 1.1 | 1.1 | 1.0 | 1.4 |
• | T “Group I” versus N “Group I” | −1.3 | −1.5 | −1.5 | −1.1 | 1.5 | 1.5 | 2.0 | 1.3 | 1.8 | 1.6 | 1.6 | 1.5 | 1.6 |
• | T “Group II” versus N “Group II” | −1.3 | −1.2 | −1.2 | −1.3 | 1.5 | 1.2 | 3.2 | 1.1 | 2.8 | 1.8 | 1.22 | 1.82 | 1.75 |
Groups | Spots | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
383 | 397 | 398 | 400 | 393 | 396 | 402 | 403 | 404 | 405 | 501 | 526 | 548 | ||
• | “Tumor staging” Group II (nr = 19) versus Group I (nr = 9) | −1.3 | 1.0 | −1.2 | −1.3 | 1.4 | 1.2 | 3.2 | 1.1 | 2.8 | 1.8 | 1.2 | 1.8 | 1.8 |
• | “Anatomical subsites” Antrum (nr = 8) versus. corpus (nr = 18) | −1.0 | −1.9 | −2.0 | 1.4 | 1.7 | 1.7 | 3.4 | 1.9 | 2.8 | 2.3 | 1.9 | −1.5 | −1.7 |
• | “Histological subtypes” Diffuse (nr = 18) versus intestinal (nr = 7) | −1.1 | 1.0 | −1.3 | −1.3 | −1.3 | −1.1 | −2.1 | 1.0 | −2 | −1.7 | 3.0 | 1.3 | −1.1 |
• | “Age” >50 years (nr = 19) versus <50 years (nr = 7) | 1.1 | 1.4 | 1.2 | 1.1 | 1.1 | −1.1 | 3.6 | −1.0 | 1.8 | 2.0 | −1.1 | 1.1 | 1.2 |
• | “Sex” Female (nr = 15) versus male (nr = 13) | 1.3 | 2.0 | 1.8 | 1.3 | −1.0 | 1.0 | −1.0 | −1.1 | 1.2 | 1.1 | −2 | −1.3 | 1.0 |
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Repetto, O.; Maiero, S.; Magris, R.; Miolo, G.; Cozzi, M.R.; Steffan, A.; Canzonieri, V.; Cannizzaro, R.; De Re, V. Quantitative Proteomic Approach Targeted to Fibrinogen β Chain in Tissue Gastric Carcinoma. Int. J. Mol. Sci. 2018, 19, 759. https://doi.org/10.3390/ijms19030759
Repetto O, Maiero S, Magris R, Miolo G, Cozzi MR, Steffan A, Canzonieri V, Cannizzaro R, De Re V. Quantitative Proteomic Approach Targeted to Fibrinogen β Chain in Tissue Gastric Carcinoma. International Journal of Molecular Sciences. 2018; 19(3):759. https://doi.org/10.3390/ijms19030759
Chicago/Turabian StyleRepetto, Ombretta, Stefania Maiero, Raffaella Magris, Gianmaria Miolo, Maria Rita Cozzi, Agostino Steffan, Vincenzo Canzonieri, Renato Cannizzaro, and Valli De Re. 2018. "Quantitative Proteomic Approach Targeted to Fibrinogen β Chain in Tissue Gastric Carcinoma" International Journal of Molecular Sciences 19, no. 3: 759. https://doi.org/10.3390/ijms19030759