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

Abstract

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.

Keywords: Alpha-1-antitrypsin; Biomarkers; Estrogen receptor positive and negative breast cancer; Fourier transform ion cyclotron resonance mass spectrometry; Proteomics; Two-dimensional gel electrophoresis.

Figure 1

Western Blot to verify the presence of estrogen receptor in breast tissue samples. Western blot of 25 µg of ductal breast cancer tissue samples (1, 2, & 3) for estrogen receptor alpha are shown on the right side of the figure. The left side represents those samples in Ponseau S staining that shows the total loading of protein. Sample 1 is known to be ER+ from its pathology report.

Figure 2

2-DE gel scans presenting glycoproteins versus total proteins. Double staining 2-DE gels of human breast tissue samples with estrogen receptor alpha positive (ER+), estrogen receptor alpha negative (ER-) samples, and their treated N-deglycosylated samples (ER+D) and (ER-D). Double staining of 2-DE gels utilized Pro-Q Emerald 300 glycoprotein staining C and D followed by Sypro Ruby total protein gel staining A and B. 125 µg of total lysate was loaded on 4-7 pH ranged 10% SDS-PAGE gels. The sugar cane molecular weight ladder provided in the Multiplexed Proteomics Glycoprotein Gel Stain Kit is presented at the left of each 2-DE gel.

Figure 3

Glycoprotein and total protein spot count in ER+ versus ER- samples. The mean number of total protein and glycoprotein in ER+ and ER- 2-DE gels was obtained from ImageQuant TL. Spot count data were collected from 12 iterations of 2-DE gels for ER+ and ER- total proteins and from 6 iterations of ER+ and ER- glycoproteins. Six out of 12 iterations had double staining for glycoproteins and total proteins. Error bars represent a 95% confidence interval calculated from the standard deviation of each sample.

Figure 4

Progenesis SameSpot v3.3 software results for the expression level of spots 1 through 10 after Sypro Ruby (total protein) and ProQ Emerald 300 glycoprotein staining of the ER+, ER+D, ER-, and ER-D gels. ER represents the estrogen receptor. ER+D represent the N-deglycosylated estrogen receptor positive sample. Error bars represent a 95% confidence interval calculated by the Progenesis SameSpot v3.3 software from the normalized-to-background peak volume and peak height of each spot/protein from each 2-DE gel.

Figure 5

Western blot for A1AT, A1A, SOD2, and CaM in: normal (well 1), ER+ PR+ (well 2), ER+ PR weakly + (well 3), ER+ PR- (well 4) (whose corresponding normal is in well 1), and ER- PR- (well 5) breast tissue samples. B-actin was used as a loading control. 25 µg of each sample was loaded onto a 10% SDS-PAGE gel. Novex® Sharp Pre-stained Protein Standard (Invitrogen) was used as the molecular weight marker (Mr).

Figure 6

Western blot for A1AT, SOD2, and CaM in: normal (well 1), ER+ PR+ breast cancer tissue (well 2) whose corresponding normal is in well 1, normal (well 3), ER+ PR- breast cancer tissue (well 4) whose corresponding normal is in well 3, normal (well 5), and ER- PR- breast cancer tissue (well 6) whose corresponding normal is in well 5. B-actin was used as a loading control. 25 µg of each sample was loaded on a 10% SDS-PAGE gel. Same molecular weight marker used in Figure 5.

Figure 7

A1AT, A1A, SOD2, and CaM's expression level in breast cancer tissues. There is no significant difference among the expression level of any of these proteins in any of the presented cancer samples. The relative protein intensity was produced by Adobe Photoshop 6.0 by comparing their absolute intensity to B-actins from all Western blot trials. The absolute intensity for each protein in each sample was calculated by multiplying the mean and pixels of the band. Error bars represent a 95% confidence interval calculated from the standard deviation of each sample. ER+PR+ represents estrogen receptor and progesterone receptor positive breast cancer tissue sample. ER+PR~+ represents a breast cancer tissue sample with ER+ and PR weakly positive.

Figure 8

Western blot to verify any difference in bands is due to a glycosylation difference. Western blots for A1AT, A1A, SOD2, and CaM were performed with 10% SDS-PAGE gel. With a glycoprotein isolation kit WGA, 25 µg of accumulated glycoproteins from the breast cancer tissue samples ER+ PR+ (well 1), ER+ PR- (well 2), ER- PR- (well 3), ER- PR- N-deglycosylated (well 4) (same ER- PR- sample but N-deglycosylated), and ER- PR- control (well 5) (same ER- PR- sample used treated with water instead of PNGase F enzyme) were loaded onto the Western blot. Their flow-through, containing the rest of the proteins in each sample other than the WGA lectin-attached glycoproteins, was loaded onto the same Western blot in wells 6 to 10. As a negative control, bovine serum albumin was loaded in well 11. Presented is the same Western blot at low and high exposure time to the nitrocellulose membrane for A1AT (A) & (B), A1A (C) & (D), and SOD2 (E) & (F), respectively, whereas only high nitrocellulose exposure of CaM and B-actin are presented as (G) and (H). The same molecular weight marker (kDa) was used as in Figure 5. The * in (B) points to the ~69 kDa band on its left.