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. 2012 Aug;28(2):429-38.
doi: 10.3892/or.2012.1849. Epub 2012 Jun 1.

Comparison of Tear Protein Levels in Breast Cancer Patients and Healthy Controls Using a De Novo Proteomic Approach

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Free PMC article

Comparison of Tear Protein Levels in Breast Cancer Patients and Healthy Controls Using a De Novo Proteomic Approach

Daniel Böhm et al. Oncol Rep. .
Free PMC article

Abstract

Noninvasive biomarkers are urgently needed for early detection of breast cancer since the risk of recurrence, morbidity and mortality are closely related to disease stage at the time of primary surgery. In the past decade, many proteomics-based approaches were developed that utilize the protein profiling of human body fluids or identification of putative biomarkers to obtain more knowledge on the effects of cancer emergence and progression. Herein, we report on an analysis of proteins in the tear fluid from breast carcinoma patients and healthy women using a de novo proteomic approach and 25 mixed samples from each group. This study included 25 patients with primary invasive breast carcinoma and 25 age-matched healthy controls. We performed a MALDI-TOF-TOF-driven semi-quantitative comparison of tear protein levels in cancer (CA) and control (CTRL) using a de novo approach in pooled samples. Over 150 proteins in the tear fluid of CTRL and CA were identified. Using an in-house-developed algorithm we found more than 20 proteins distinctly upregulated or downregulated in the CTRL and CA groups. We identified several proteins that had modified expression in breast cancer patients. These proteins are involved in host immune system pathways (e.g., C1Q1 or S100A8) and different metabolic cascades (ALDH3A or TPI). Further validation of the results in an independent population combined with individual protein profiling of participants is needed to confirm the specificity of our findings and may lead to a better understanding of the pathological mechanism of breast cancer.

Figures

Figure 1
Figure 1
The stained tear samples from CTRL and CA. As an example, the composed grid is shown on the CTRL lane. The lane M shows the protein standard (SeeBlue® Plus2 Pre-Stained Standard Invitrogen, Darmstadt, Germany).
Figure 2
Figure 2
Mass spectra of the digested and purified peptides from the CTRL and CA groups. The molecular weight of the digested peptides is shown on the x-axis; while, the y-axis represents the intensity of the signals obtained. The spectra show the peptides derived from the first gel fraction in both groups. Spectra 1 and 2 show the peptides eluted with 10% acetonitrile (ACN). Spectra 3 and 4 demonstrate the peptides after elution with 15% ACN. Spectra 5 and 6 represent peptides from the elution step with 30% ACN. All spectra show the reproducible high intensity of signals. The mass spectra show appropriate differences in the peptide patterns according to the CTRL and CA pools.
Figure 3
Figure 3
Network of identified proteins in the tear fluid of CA and CTRL. Using the software Cytoscape 2.7.0, we constructed a network of the merged proteins identified in both groups. In this overview, the proteins are clustered according to the molecular functions. The ontology file GO_Molecular_Function was used. Other criteria were: hypergeometric statistic test, accessing overrepresented categories, and significance level of 0.05. The circled areas summarize the identified proteins and their molecular functions as follows: Yellow circle: histone modification, transferase activity; blue circle: binding of DNA, lipids and proteins, transcription activation; red circle: transmembrane transport activity; and green circle: catalytic activity. The yellow color on the nodes indicates a higher number of the assigned proteins.
Figure 4
Figure 4
Using the software, STRING, we constructed an overview of the proteins that were at least 2-fold differently regulated in CTRL and CA. The appropriate gene names are abbreviated. The arrows show the increase (red arrow) or decrease (blue arrow) of the proteins in CA. Some of the known interactions of the proteins are shown with connection lines. The thickness of the lines shows how strong the interactions are.

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