Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+) and Negative (ER-) Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry.

Breast cancer is the second most fatal cancer in American women. To increase the life expectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+) tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER-) tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining) and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples.

Differential phosphopeptide expression in a benign breast tissue, and triple-negative primary and metastatic breast cancer tissues from the same African-American woman by LC-LTQ/FT-ICR mass spectrometry.

African-American women have a higher risk for developing triple-negative breast cancer (TNBC). Lacking the expression of receptors for estrogen and progesterone, and without human epidermal growth factor 2 receptor gene amplification, TNBC is a very aggressive type of breast cancer with a high likelihood of metastasis and recurrence. Specific therapeutic targets for this aggressive disease remain to be identified. Phosphorylation, a post-translational modification that adds one or more phosphate groups to a protein, plays a key role in the activation and deactivation of a protein's cellular function. Here, we report the first systematic phosphoproteomic analysis of a benign breast tissue, a primary breast cancer tissue, and a metastatic breast cancer tissue from the same African-American woman. Differential phosphoprotein levels were measured with reversed-phase nano-liquid chromatography coupled to a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FT-ICR MS). Five proteins were found to be highly phosphorylated in the metastatic site whereas six proteins were highly phosphorylated in the cancer site of the TNBC patient. Identified phosphoproteins are known to be involved in breast cancer signal transduction pathways and these results may identify new diagnostic and therapeutic targets for TNBC.

Prefractionation enhances loading capacity and identification of basic proteins from human breast cancer tissues.

Many basic proteins (pI>7) and putative disease biomarkers are not identified using conventional proteomic methods. This study applied a new method to improve the identification of such proteins. Prefractionated basic proteins were compared with total tissue lysates from human ductal carcinoma in situ tissue loaded on basic immobilized pH gradient strips prior to two-dimensional gel electrophoresis (2-DE). Extraction of alkaline proteins was achieved in less than 20 min using a chromatofocusing resin and two buffers in a microcentrifuge tube. Prefractionation showed improved resolution and visualization of low-abundance proteins on 2-DE gels, allowing proteins to be excised, accumulated, trypsin-digested, and identified by liquid chromatography-tandem mass spectrometry. Proteins identified in the prefractionated samples had a higher number of peptides and three times the number of unique basic proteins when compared with total lysates. Low-molecular-weight (LMW, <26kDa) unique alkaline proteins comprise 75% of those identified in prefractionated samples compared with 25% identified in total lysates, representing a 9-fold increase of LMW proteins due to prefractionation. Prefractionation ultimately increases loading capacity of samples onto the 2-DE gel and leads to better resolution, visualization, and identification of proteins with pI values greater than 7.

Alteration in protein expression in estrogen receptor alpha-negative human breast cancer tissues indicates a malignant and metastatic phenotype.

Ductal carcinoma in situ (DCIS) represents the earliest identifiable breast cancer lesion. Disruption of the myoepithelial cell layer and basement membrane is a prerequisite for DCIS to initiate invasion into the stroma. The majority of epithelial cells overlying a focally-disrupted myoepithelial cell layer are estrogen receptor-alpha negative (ER(-)); however, adjacent cells within the same duct confined by an intact myoepithelial cell layer express high levels of ER. These ER (+) and ER (-) cells were microdissected from the same ducts of breast cancer patients. Differential proteins expressed by ER(+) and ER(-) cells were identified using two-dimensional gel electrophoresis followed by mass spectrometry and Western blot analysis. ER(-) cells express lower levels of superoxide dismutase, RalA binding protein, galectin-1, uridine phosphorylase 2, cellular retinoic acid-binding protein 1, S100 calcium binding protein A11, and nucleoside diphosphate kinase A or non-metastasis protein 23-H1 (nm23-H1). The upregulated protein, Rho GDP-dissociation inhibitor 1 alpha, may induce chemotherapy resistance. The significant findings are that the microdissected ER(-) cells express 12.6 times less cellular retinoic acid-binding protein 1, a protein involved in cellular differentiation, and 4.1 times less nucleoside diphosphate kinase A or nm23-H1, a metastasis suppressor, and express fewer proteins than adjacent ER(+) cells. The collective role of the alterations of protein expression in ER(-) cells may be to promote a more malignant phenotype than adjacent ER(+) cells, including a decreased ability to undergo apoptosis and differentiation, and an increased potential to damage DNA, metastasize, and resist to chemotherapy.

Methodology and applications of disease biomarker identification in human serum.

Biomarkers are biomolecules that serve as indicators of biological and pathological processes, or physiological and pharmacological responses to a drug treatment. Because of the high abundance of albumin and heterogeneity of plasma lipoproteins and glycoproteins, biomarkers are difficult to identify in human serum. Due to the clinical significance the identification of disease biomarkers in serum holds great promise for personalized medicine, especially for disease diagnosis and prognosis. This review summarizes some common and emerging proteomics techniques utilized in the separation of serum samples and identification of disease signatures. The practical application of each protein separation or identification technique is analyzed using specific examples. Biomarkers of cancers of prostate, breast, ovary, and lung in human serum have been reviewed, as well as those of heart disease, arthritis, asthma, and cystic fibrosis. Despite the advancement of technology few biomarkers have been approved by the Food and Drug Administration for disease diagnosis and prognosis due to the complexity of structure and function of protein biomarkers and lack of high sensitivity, specificity, and reproducibility for those putative biomarkers. The combination of different types of technologies and statistical analysis may provide more effective methods to identify and validate new disease biomarkers in blood.