Detection and identification of potential biomarkers of breast cancer

Abstract

Purpose: Noninvasive and convenient biomarkers for early diagnosis of breast cancer remain an urgent need. The aim of this study was to discover and identify potential protein biomarkers specific for breast cancer.

Methods: Two hundred and eighty-two (282) serum samples with 124 breast cancer and 158 controls were randomly divided into a training set and a blind-testing set. Serum proteomic profiles were analyzed using SELDI-TOF-MS. Candidate biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays and western blot technique.

Results: A total of 3 peaks (m/z with 6,630, 8,139 and 8,942 Da) were screened out by support vector machine to construct the classification model with high discriminatory power in the training set. The sensitivity and specificity of the model were 96.45 and 94.87%, respectively, in the blind-testing set. The candidate biomarker with m/z of 6,630 Da was found to be down-regulated in breast cancer patients, and was identified as apolipoprotein C-I. Another two candidate biomarkers (8,139, 8,942 Da) were found up-regulated in breast cancer and identified as C-terminal-truncated form of C3a and complement component C3a, respectively. In addition, the level of apolipoprotein C-I progressively decreased with the clinical stages I, II, III and IV, and the expression of C-terminal-truncated form of C3a and complement component C3a gradually increased in higher stages.

Conclusions: We have identified a set of biomarkers that could discriminate breast cancer from non-cancer controls. An efficient strategy, including SELDI-TOF-MS analysis, HPLC purification, MALDI-TOF-MS trace and LC-MS/MS identification, has been proved very successful.

Fig. 1

A representative spectrum of SELDI-TOF-MS analysis of sera from breast cancer patients and healthy controls. Differentially expressed proteins with potential diagnostic significance are arrowed. Top group denotes sera from patients with breast cancer, in which 8,139 and 8,942 m/z were over-expressed. Bottom group denotes sera from healthy individuals, in which 6,630 m/z was up-regulated

Fig. 2

A representative spectrum of SELDI-TOF-MS analysis of sera from different stages of breast cancer patients and non-cancer controls. The level of 8,139, 8,942 Da proteins progressively increased with the clinical stages I, II, III and IV, and the expression of 6,630 Da protein gradually decreased with the stage increasing

Fig. 3

MALDI-TOF-MS spectra of three purified potential protein markers

Fig. 4

Results of the identification of protein (8,942 Da) by LC-MS/MS. a Chromatogram of peptide mixture; b, c MS/MS spectra of two peptides

Fig. 5

Representative spectra from ProteinChip array with immobilized antibodies against apolipoprotein C-I (b), complement component C3a (c) for breast cancer patients and healthy individuals and representative spectra of the negative control (nonspecific mouse IgG; a)

Fig. 6

a Western blots of apolipoprotein C-I (APO CI) in sera from breast cancer and non-cancer control. b The expression of APO CI in breast cancer patients is lower than non-cancer controls. In comparison with the control group, there is significant difference between them (*t = −22.951, P < 0.001)

Fig. 7

a Western blots of C-terminal-truncated form of C3a in sera from breast cancer and non-cancer control. b The expression of C-terminal-truncated form of C3a in breast cancer patients is higher than non-cancer controls. In comparison with the control group, there is significant difference between them (*t = 13.684, P < 0.001)

Fig. 8

a Western blots of complement component C3a in sera from breast cancer and non-cancer control. b The expression of C3a in breast cancer patients is higher than non-cancer controls. In comparison with the control group, there is significant difference between them (*t = 13.974, P < 0.001)