Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer
Abstract
:1. Introduction
2. Results
2.1. Biomarker Discovery
2D Gel-Based Proteomic Profiling of Normal and Matched CRC Tissue Samples
2.2. Biomarker Exploratory Phase
2.2.1. Selection of Candidate Proteins
2.2.2. Application to Human Plasma Samples for Verification of Clinical Utility
3. Discussion
4. Materials and Methods
4.1. Biological Material
4.1.1. Patient Sample Collection and Handling for 2D PAGE Analysis
4.1.2. Patient Plasma Sample Collection and Handling for Protein Extension Analysis
4.2. Two-Dimensional PAGE
4.3. Mass Spectrometry
4.3.1. In-Gel Digestion of Proteins Separated by Gel Electrophoresis
4.3.2. Sample Preparation
4.3.3. Peptide Mass Mapping by MALDI-MS
4.4. Indirect Immunofluorescence Analysis
4.5. Proximity Extension Analysis (PEA) Validation Analysis
4.5.1. Quality Criteria for Antibody Assessment
4.5.2. Establishment of the PEA Probes
4.5.3. Proximity Extension Assay (PEA)
4.5.4. Real-Time qPCR
4.6. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Identity | Apparent Mw (kDa) | Apparent pl | Sample Frequency a (Dukes’) | ||||
---|---|---|---|---|---|---|---|
A | B | C | D | All Tumors | |||
40S Ribosomal protein s10 | 17 | 10.3 | 0/1 | 4/14 | 2/9 | 1/7 | 7/31 |
Annexin II | 38.5 | 7.9 | 1/1 | 7/14 | 3/9 | 5/7 | 16/31 |
Annexin VII | 37.5 | 5.6 | 1/2 | 1/4 | - | 2/2 | 7/12 |
Calreticulin b | 60.7 | 4.2 | 2/2 | 4/4 | - | 2/2 | 11/12 |
Calreticulin b | 60.7 | 4.2 | 2/2 | 4/4 | - | 2/2 | 11/12 |
Catalase | 66 | 7.9 | 0/1 | 7/14 | 2/7 | 2/7 | 11/29 |
Cofilin | 19 | 8.2 | 2/3 | 9/12 | 4/8 | 7/8 | 22/31 |
Cyclophilin A b | 18.2 | 8 | 1/1 | 12/14 | 6/9 | 6/7 | 25/31 |
Cyclophilin A b | 18.1 | 8.3 | 0/3 | 11/11 | 7/9 | 7/8 | 25/28 |
Cyclophilin B b | 19.8 | 10.2 | 1/1 | 7/14 | 6/9 | 5/7 | 19/31 |
Cyclophilin B b | 19.1 | 10.2 | 1/1 | 9/14 | 5/9 | 3/7 | 18/31 |
EF-1ϒ | 33 | 4.3 | 1/2 | 3/4 | - | 2/2 | 8/10 |
Eukaryotic translation initiation factor 5A-1 (eIF-4D) | 16 | 4.9 | 2/2 | 3/4 | - | 2/2 | 9/11 |
Elongation factor 1 alpha (EF-1α) | 19.7 | 9.9 | 4/6 | 16/19 | 16/18 | 7/8 | 43/51 |
Eukaryotic translation elongation factor 2 (EF-2) | 94.3 | 6.4 | 1/2 | 3/3 | - | 1/1 | 8/9 |
F1 ATPase, alpha-subunit | 53.8 | 7.6 | 0/1 | 4/14 | 4/9 | 2/7 | 10/31 |
Fructose biphosphate aldolase A | 43.4 | 8.8 | 1/3 | 5/11 | 4/8 | 4/8 | 14/30 |
Fumarate hydrotase | 44.2 | 7.9 | 1/1 | 11/14 | 6/9 | 5/7 | 23/31 |
FUSE binding protein (FBP, myc far upstream element-binding protein) | 67.5 | 7.6 | 2/3 | 10/12 | 9/10 | 7/7 | 28/32 |
FUSE binding protein 2 (FBP2, KH-type splicing regulatory protein KSRP mRNA) | 75 | 7.5 | 2/3 | 11/12 | 6/10 | 6/7 | 25/32 |
FUSE binding protein 3 (FBP3) | 63.4 | 8.2 | 0/1 | 10/14 | 5/9 | 3/7 | 18/31 |
Galectin-3 | 26.1 | 8.6 | 1/1 | 6/14 | 4/9 | 5/7 | 16/31 |
Glutamate dehydrogenase | 53.4 | 7.9 | 1/1 | 11/14 | 9/9 | 6/7 | 27/31 |
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) b | 19.1 | 8.9 | 4/6 | 16/19 | 16/18 | 7/8 | 43/51 |
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) b | 19.9 | 8.9 | 4/6 | 16/19 | 16/18 | 7/8 | 43/51 |
Endoplasmic reticulum chaperone BiP (GRP78) | 75.5 | 4.9 | - | 2/4 | - | 1/2 | 6/10 |
Glutathione S-transferase P (GSTπ) | 25.4 | 5.5 | 1/2 | 2/4 | 1/2 | 1/2 | 10/13 |
Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) | 38.9 | 8.3 | 1/1 | 6/14 | 4/9 | 4/7 | 15/31 |
Homo sapiens DNA-binding protein (CROC-1B) | 19 | 7.1 | 0/1 | 11/14 | 6/9 | 4/7 | 21/31 |
HSP47 (collagen-binding protein 2) | 48.6 | 9.3 | 2/3 | 12/12 | 9/9 | 7/8 | 30/32 |
Heat shock protein 90 (Hsp90) | 89.5 | 4.9 | 2/2 | 1/4 | - | 2/2 | 6/11 |
IG gamma-1 chain C region | 67 | 7.6 | 1/1 | 14/14 | 7/9 | 7/7 | 29/31 |
Isocitrate dehydrogenase | 46.8 | 7 | 0/1 | 7/14 | 2/7 | 2/7 | 11/29 |
Keratin 18 | 43.6 | 5.3 | 3/3 | 3/4 | 1/1 | 1/2 | 9/12 |
Ku antigen (86 kDa) | 85.6 | 5.7 | 3/3 | 2/4 | 1/2 | 8/11 | |
Leukocyte elastase inhibitor | 17 | 7.3 | 0/1 | 5/14 | 5/9 | 2/7 | 12/31 |
Maspin | 38.7 | 5.8 | 3/3 | 3/3 | - | 0/2 | 8/11 |
Monocarboxylate transporter 1 (MCT-1) | 19.1 | 10.2 | 1/1 | 2/14 | 5/9 | 4/7 | 17/31 |
Nucleoside diphosphate kinase B (NDK B) | 19 | 8.7 | 2/3 | 11/12 | 9/10 | 5/7 | 27/32 |
Proteasome activator complex subunit 1 (PA28α) | 30.4 | 5.8 | 3/3 | 4/4 | - | 1/2 | 10/12 |
Proliferating cell nuclear antigen (PCNA) | 37.4 | 4.4 | 2/3 | 2/3 | 2/2 | 2/2 | 9/12 |
Peroxiredoxin-1 | 22 | 8.2 | 2/3 | 13/13 | 9/10 | 8/8 | 32/34 |
Phosphoglycerate kinase 1 | 43.6 | 8.2 | 1/1 | 9/14 | 4/7 | 6/7 | 20/29 |
Porin 2 | 35.3 | 7.9 | 2/3 | 9/11 | 7/10 | 4/8 | 22/32 |
Profilin-1 | 15.8 | 8.4 | 1/1 | 10/14 | 7/9 | 5/7 | 23/31 |
Proteasome | 26.9 | 5.6 | - | 2/4 | - | 1/2 | 6/12 |
Proteasome C3 | 26.5 | 7.2 | 1/1 | 6/14 | 6/9 | 4/7 | 17/31 |
Proteasome Ϛ | 28.1 | 4.4 | 2/3 | 2/4 | 1/1 | 2/2 | 11/14 |
Protein disulfide isomerase (PDI) | 56.2 | 4.6 | 2/2 | 4/4 | 1/1 | 2/2 | 11/12 |
Protein disulfide isomerase ER 60 precursor | 56.5 | 5.7 | 2/2 | - | - | 1/2 | 9/11 |
Putative secreted protein XAG2 | 17.3 | 10 | 1/1 | 10/14 | 4/9 | 3/7 | 18/31 |
Receptor of activated protein C kinase 1 (Rack1) | 32.5 | 8.3 | 3/3 | 9/11 | 6/10 | 3/8 | 21/32 |
Raf kinase inhibitor protein (RKIP) | 23 | 8.2 | 1/1 | 11/14 | 4/9 | 7/7 | 23/31 |
Smooth muscle protein 22-alpha (SM-22α) | 24.9 | 8.3 | 1/3 | 8/10 | 2/6 | 6/8 | 17/27 |
Sorbitol dehydrogenase | 42.4 | 8.3 | 1/1 | 10/14 | 7/9 | 3/7 | 21/31 |
Splicing factor SF2 p32 subunit | 35.5 | 4.2 | 1/1 | 4/4 | - | 2/2 | 10/10 |
Thyroid hormone receptor-interacting protein 1 (26S protease regulatory subunit 8) | 46.8 | 7.1 | 1/1 | 12/14 | 6/7 | 6/7 | 26/31 |
Translationally controlled tumor protein (TCTP) | 24.2 | 4.6 | 0/2 | 2/4 | 0/1 | 2/2 | 6/12 |
Triosephosphate isomerase | 28.3 | 7.3 | 1/1 | 11/14 | 7/9 | 5/7 | 24/31 |
Tropomyosin-3 (tm3) | 33.7 | 4.4 | 2/3 | 2/4 | - | 2/4 | 8/10 |
Vinculin (metavinculin) | 65 | 7.5 | 1/1 | 13/14 | 4/9 | 7/7 | 25/31 |
α-Enolase | 49.2 | 7.5 | 1/1 | 11/14 | 4/7 | 5/7 | 21/29 |
Protein Name | Symbol | UniProtKB | Deregulation Frequency c |
---|---|---|---|
78 kDa glucose-regulated protein a,b | Bip/Grp78/HspA5 | P11021 | 6/10 |
α-Enolase a | Eno1 | P06733 | 21/29 |
Catalase a,b | Cat | P04040 | 11/29 |
Cyclophilin B a,b | Scylp/PPIase B/PPIB | P23284 | 18/31 |
Eukaryotic translation elongation factor 2 a | eEF2/EF-2 | P13639 | 8/9 |
Galectin-3 a,b | Gal-3 | P17931 | 16/31 |
Peroxiredoxin-1 a,b | Prdx1/Nkef-A | Q06830 | 32/34 |
Profilin-1 a | Pfn1 | P07737 | 23/31 |
Receptor for activated C kinase 1 a,b | Rack1 | P38011 | 21/32 |
Tropomyosin alpha-3 chain a,b | Tpm3/tm3 | P06753 | 8/10 |
Protein | Expression in CRC Samples | HPA Concordance (Reference Antibody) |
---|---|---|
Grp78 | Expression in tumor cells, with samples varying from weak to very strong expression | Yes (HPA038845) |
Eno1 | Expression in tumor cells, with samples varying from not detected to strong expression | Yes (CAB018614) |
Cat | Some samples showed low to moderate expression in tumor cells | No (HPA051282) |
PPIB | Expression in tumor cells, with samples varying from not detected to strong expression | Yes (HPA012720) |
eEF2 | Ubiquitous strong expression in tumor cells | Yes (HPA040534) |
Gal-3 | Expression in tumor cells, with samples varying from weak to very strong expression | Yes (HPA003162) |
Prdx1 | Expression in tumor cells, with samples varying from weak to very strong expression | Yes (CAB004682) |
Pfn1 | Some samples showed low to moderate expression in tumor cells | Yes (CAB037134) |
Rack1 a | Medium to strong expression in tumor cells, with samples varying from not detected to strong expression | Yes (CAB004288) |
Tpm3 b | Strong expression in skeletal muscle and endothelial cells. Tumor cells showed low to moderate expression in tumor cells | Yes (HPA009066) |
Dukes’ Stage | Loc. 1 | Loc. 2 | Loc. 3 | Loc. 4 | Loc. 5 | Loc. 6 | Loc. 7 | Loc. 8 | Loc. 9 | Loc. 10 | Loc. 11 | Total |
---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 2 | 2 | 1 | 0 | 0 | 1 | 2 | 2 | 1 | 3 | 1 | 15 |
B | 8 | 6 | 0 | 1 | 3 | 1 | 14 | 2 | 7 | 7 | 0 | 49 |
C | 6 | 2 | 1 | 1 | 1 | 1 | 10 | 8 | 1 | 17 | 0 | 48 |
D | 1 | 0 | 1 | 0 | 0 | 1 | 8 | 1 | 2 | 2 | 0 | 16 |
Total | 17 | 10 | 3 | 2 | 4 | 4 | 34 | 13 | 11 | 29 | 1 |
Dukes’ Stage | Loc. 1 | Loc. 2 | Loc. 3 | Loc. 4 | Loc. 5 | Loc. 6 | Loc. 7 | Loc. 8 | Loc. 9 | Loc. 10 | Loc. 11 | Total |
---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 2 | 2 | 1 | 0 | 0 | 2 | 1 | 2 | 1 | 4 | 1 | 16 |
B | 9 | 5 | 0 | 1 | 3 | 1 | 16 | 2 | 7 | 7 | 0 | 51 |
C | 7 | 3 | 1 | 1 | 2 | 1 | 9 | 9 | 2 | 18 | 1 | 54 |
D | 1 | 0 | 1 | 0 | 0 | 1 | 8 | 1 | 4 | 2 | 0 | 18 |
No cancer | 3 | 1 | 0 | 2 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 9 |
Total | 22 | 11 | 3 | 4 | 5 | 5 | 35 | 15 | 15 | 31 | 2 |
Subjects, n (%) | ||
---|---|---|
Gender | ||
Female | 35 (50) | |
Male | 35 (50) | |
Age group | ||
40–49 | 3 (4) | |
50–59 | 9 (13) | |
60–69 | 17 (24) | |
70–79 | 22 (31) | |
80–89 | 18 (26) | |
90–99 | 1 (1) | |
Stage | ||
TNM | AJCC * | |
T1, T2-N0-M0 | I | 7 (10) |
T3-N0-M0/T4-N0-M0 | II | 29 (41) |
T1, T2-N1-M0/T3, T4-N1-M0 | III | 15 (21) |
Any T-N2-M0/Any T-Any N-M1 | IV | 14 (20) |
Not specified | Not specified | 5 (7) |
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Thorsen, S.F.; Gromova, I.; Christensen, I.J.; Fredriksson, S.; Andersen, C.L.; Nielsen, H.J.; Stenvang, J.; Moreira, J.M.A. Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer. Int. J. Mol. Sci. 2019, 20, 6082. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20236082
Thorsen SF, Gromova I, Christensen IJ, Fredriksson S, Andersen CL, Nielsen HJ, Stenvang J, Moreira JMA. Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer. International Journal of Molecular Sciences. 2019; 20(23):6082. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20236082
Chicago/Turabian StyleThorsen, Stine F., Irina Gromova, Ib J. Christensen, Simon Fredriksson, Claus L. Andersen, Hans J. Nielsen, Jan Stenvang, and José M.A. Moreira. 2019. "Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer" International Journal of Molecular Sciences 20, no. 23: 6082. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20236082