Targeted glycoproteomic identification of biomarkers for human breast carcinoma

Abstract

Glycosylation is a dynamic post-translational modification that changes during the development and progression of various malignancies. During the oncogenesis of breast carcinoma, the glycosyltransferase known as N-acetylglucosaminyltransferase Va (GnT-Va) transcript levels and activity are increased due to activated oncogenic signaling pathways. Elevated GnT-V levels leads to increased beta(1,6)-branched N-linked glycan structures on glycoproteins that can be measured using a specific carbohydrate binding protein or lectin known as L-PHA. L-PHA does not bind to nondiseased breast epithelial cells, but during the progression to invasive carcinoma, cells show a progressive increase in L-PHA binding. We have developed a procedure for intact protein L-PHA-affinity enrichment, followed by nanospray ionization mass spectrometry (NSI-MS/MS), to identify potential biomarkers for breast carcinoma. We identified L-PHA reactive glycoproteins from matched normal (nondiseased) and malignant tissue isolated from patients with invasive ductal breast carcinoma. Comparison analysis of the data identified 34 proteins that were enriched by L-PHA fractionation in tumor relative to normal tissue for at least 2 cases of ductal invasive breast carcinoma. Of these 34 L-PHA tumor enriched proteins, 12 are common to all 4 matched cases analyzed. These results indicate that lectin enrichment strategies targeting a particular glycan change associated with malignancy can be an effective method of identifying potential biomarkers for breast carcinoma.

Figure 1

(A) A tetra-antennary N-linked oligosaccharide showing the GnT-V β(1,6) GlcNAc addition that leads to the formation of polylactosamine structures. The L-PHA recognition site is circled. (B) Schematic flow diagram for the L-PHA enrichment protocol.

Figure 2

Enrichment of tetra-antennary glycans extended with N-acetyllactosamine in tumor and adjacent normal tissue from case 2417. The four indicated N-linked glycans (1–4) were detected by NSI-MS/MS. In the profiles shown at the right of each glycan, the MS/MS spectra associated with the TIM scan for the indicated tissue were filtered to present the detected signal intensity of a signature tetrasaccharide fragment (Hex-HexNAc-Hex-HexNAc). The presence of this fragment indicates the detection of a glycan extended with at least two N-acetyllactosamine repeats at a scan time which predicts the m/z ratio for the parent ion. For reference, the scan time for specifc m/z values is indicated by arrows in each filtered profile. The shading and shapes for the glycan structures reflect standard nomenclature adopted by the Consortium for Functional Glycomics (CFG; GlcNAc, blue square; Gal, yellow circle, Man, green circle; Fuc, red triangle, NeuAc, pink diamond).

Figure 3

Functional annotation and distribution of the L-PHA-enriched proteins identified from breast carcinoma. (A) Biological function of proteins listed in Table 2 as annotated by DAVID 2007. (B) Cellular compartment for L-PHA-enriched proteins assigned based on GO consortium. (C) Cellular compartment of proteins identified from normal breast tissue by total MS/MS analysis assigned by GO consortium. (D) Cellular compartment of proteins identified from tumor breast tissue assigned by GO consortium.

Figure 4

Venn diagram showing the number of L-PHA-enriched proteins identified in common for each case.

Figure 5

Analysis of periostin and haptoglobin-related protein or haptoglobin by Western blot. (A) Number of peptides identified for periostin (isoform 1 and isoform 3) before L-PHA fractionation (total) and after lectin fractionation (L-PHA). (B) Precipitation of periostin using an anti-periostin antibody followed by detection using biotinylated L-PHA and streptavidin HRP (panel 1) (lower band is periostin). Total levels of periostin precipitated are confirmed by detection of the blot using anti-periostin antibody (panel 2). Reverse precipitation with L-PHA first followed by detection using an anti-periostin antibody (panel 3). Protein inputs for the L-PHA precipitations were normalized by the detection of ERK2 in 5% of the unbound fraction (panel 4). (C) Densitometry quantification of the relative increase in L-PHA reactive periostin normalized for total periostin. (D) Number of peptides identified for haptoglobin-related protein (HPR) precursor and haptoglobin (HP) by MS/MS before (total) and after L-PHA fractionation (L-PHA). (E) L-PHA precipitation followed by detection using an anti-haptoglobin antibody shows increased reactivity for the beta chains of tumor HPR/HP for cases 2417 and 2207 and, in all cases, a migratory shift to a higher molecular weight. Protein levels present in the L-PHA precipitations were determined by analysis of total haptoglobin in a 10% input blot. (F) Levels of L-PHA reactive haptoglobin were determined following densitometry analysis of L-PHA reactive HPR/HP and total HPR/HP determined from the 10% input levels.