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, 16 (1), 159-73

A Targeted Proteomic Strategy for the Measurement of Oral Cancer Candidate Biomarkers in Human Saliva

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A Targeted Proteomic Strategy for the Measurement of Oral Cancer Candidate Biomarkers in Human Saliva

Rebeca Kawahara et al. Proteomics.

Abstract

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non-invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically-derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.

Keywords: Biomedicine; Oral cancer; Saliva; Selected reaction monitoring; Skyline.

Conflict of interest statement

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Experimental workflow of SRM assay development using empirical measurement for selection of proteotypic peptides.
Figure 2
Figure 2. Analytical assay performance using CFB heavy peptides and global standards
(A) Recombinant CFB tryptic peptide intensities from 7 to 23 amino acids in length. The arrows show the proteotypic peptides measured in the saliva. The red arrows show peptides synthetized in the labeled form. B) Resulting SRM mass spectra (left, endogenous specific transitions for the different peptides corresponding to the protein used for quantification and right, corresponding heavy peptides) of VSEADSSNADWVTK and C) YGLVTYATYPK.
Figure 3
Figure 3. Proteotypic peptides identified using in vitro-synthesized protein monitored in human saliva
The relative contribution of each fragment ion to each peptide peak is displayed as different colors. The library contains the result from the recombinant, in vitro-synthetized or isolated from plasma proteins. The graph represents endogenous specific transitions for the different peptides measured in the human saliva.
Figure 4
Figure 4. Visualization for explanatory data analysis using MSstats
(A) Examples of quality control (QC) plots for Global Standards, SERPINA1 and CFB proteins. X-axis: run. Y-axis: log-intensities of transitions. B) Examples of profile plots for Global Standards, SERPINA1 and CFB proteins. Line colors indicate peptides and line types indicate transitions. C) Examples of condition plots for Global Standards, SERPINA1 and CFB proteins. X-axis: condition. Y-axis: log ratio of endogenous over reference intensities of each transition in a run. Dots indicate the mean of log ratio for each condition. Error bars have confidence intervals with 0.95 significant level for each condition. The plots visualize the differences between conditions, which are of the main biological interest.
Figure 5
Figure 5. Volcano plot of the comparison Control vs Lesion condition
X-axis: practical significance, model-based estimate of log-fold change. Y-axis: statistical significance, FDR-adjusted p-values on the negative log2 scale. The dashed line represents the FDR cutoff (default sig=0.05) and fold-change FCcutoff=1.5. A) Results at a protein level. Up regulated in OSCC (red): C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1 and SERPINA1. No regulation (black):ANXA1, SERPINE1 and Global Standards. B) Results at a peptide level. Up-regulated in OSCC (red): ANXA1 (peptides TPA, NAL); CFB (peptides YGL, VSE, DIS); C3 (peptides LMN, SSL, TFI); C4B (peptides TTN, VGD, GSF); C1R (peptide DYF); LCN2 (peptides MYA, TFV, SYP); LRG1 (peptides DLL, YLF, VAA); FAM49B (peptides MSL, VML); SERPINA1 (peptides LSI, SVL, AVL); SERPINE1 (peptide FII), SLPI (peptide CLD); TAGLN2 (peptides IQA, TLM, NVI). No regulation (black): ANXA1 (peptide ALY); SERPINE1 (peptide GMI) and C1R (peptide NLP).

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