Glycoproteomic Analysis of Malignant Ovarian Cancer Ascites Fluid Identifies Unusual Glycopeptides

Abstract

Ovarian cancer is a major cause of cancer mortality among women, largely due to late diagnosis of advanced metastatic disease. More extensive molecular analysis of metastatic ovarian cancer is needed to identify post-translational modifications of proteins, especially glycosylation that is particularly associated with metastatic disease to better understand the metastatic process and identify potential therapeutic targets. Glycoproteins in ascites fluid were enriched by affinity binding to lectins (ConA or WGA) and other affinity matrices. Separate glycomic, proteomic, and glycopeptide analyses were performed. Relative abundances of different N-glycan groups and proteins were identified from ascites fluids and a serum control. Levels of biomarkers CA125, MUC1, and fibronectin were also monitored in OC ascites samples by Western blot analysis. N-Glycan analysis of ascites fluids showed the presence of large, highly fucosylated and sialylated complex and hybrid glycans, some of which were not observed in normal serum. OC ascites glycoproteins, haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin, and alpha-1-antichymotrypsin were more abundant in OC ascites or not present in serum control samples. Further glycopeptide analysis of OC ascites identified N- and O-glycans in clusterin, hemopexin, and fibulin glycopeptides, some of which are unusual and may be important in OC metastasis.

Keywords: N-glycans; glycopeptides; glycoproteomics; malignant ascites; mass spectrometry; ovarian cancer; proteomics.

Figure 1

SDS-PAGE of WGA- and ConA-bound ascites (asc1) proteins with Western blot analysis for CA125, fibronectin, and mucins 1, 2, and 3. (A) Protein stain, (B) carbohydrate stain, (C) Western blot analysis of CA125, (D) MUC1, (E) MUC2, (F) MUC3, and (G) fibronectin. UB, unbound; B, bound; R, resin; WGA, wheat germ agglutinin; ConA, concanavalin A; asc, ascites.

Figure 2

Nano-LC Chip/TOF MS analysis of N-glycans released from proteins present in OC ascites fluid and ConA- and WGA-bound ascites. (A) Comparison of glycan chromatographs between a serum control and ascites fluid (solid line represents ascites fluid, dotted line represents serum control). (B) Comparison of chromatographs of N-glycans isolated from ascites and ConA- and WGA-bound proteins (solid line represents ascites fluid, dashed line represents ConA, and dotted line represents WGA glycans). (C) Annotated N-glycan profile of the ascites fluid analyzed by nano-LC Chip/TOF MS. (D) Pie charts of the different types (classes) of N-glycans obtained from OC ascites fluid and ConA- and WGA-bound proteins.

Figure 3

Comparative analysis of N-glycans obtained from two ascites samples Asc1 and Asc412 and a serum control sample (SS). (A) Abundant N-glycans H5N4, H5N4S1, and H5N4S2 N-glycans before and after lectin (ConA or WGA) binding in Asc1 (left), Asc412 (middle), and SS (right); (B) H5N4F1, H5N4F1S1, and H5N4F1S2 N-glycans; (C) H5N4F2S2 N-glycan; (D) H5N5, H5N5S1, and H5N5S2 N-glycans; and (E) H5N5F1, H5N5F1S1, and H5N5F1S2 N-glycans. H represents hexose; N represents N-acetyl glucosamine or GlcNAc; F represents fucose; S represents sialic acid.

Figure 4

N-Glycans in Asc1, Asc412, and SS before and after lectin binding. (A) High mannose glycans that range from H3N2 to H6N2; (B) high mannose that range from H7N2 to H10N2; (C) N-glycan groups H6N5, H6N5S1, H6N5S2, and H6N5S3 (without fucosylation); (D) N-glycan groups H6N5, H6N5S1, H6N5S2, H6N5S3 with one fucose, and H6N5F2; (E) N-glycans H6N3, H6N3S1, H6N4S1, H6N4S2, and H6N4F1S1; (F) N-glycans H7N6S1, H7N6S2, and H7N6S3 (no fucosylation); and (G) N-glycans H7N6F1S1, H7N6F1S2, and H7N6F1S3 (with fucosylation).

Figure 5

Venn diagrams comparing N-glycans from SS, Asc1, and Asc142. (A) Comparing original fluids with lectin-bound samples. (B) Comparing SS, Asc1, and Asc412 before lectin binding, with ConA binding and with WGA binding.

Figure 6

Proteins identified in ascites fluids and SS before and after lectin (ConA and WGA) lectin binding. (A) Haptoglobin, (B) fibronecctin, (C) interalpha-trypsin inhibitor heavy chains H1, H2, H3, and H4, (D) clusterin, (E) lumican, (F) ceruloplasmin, (G) fibulin, (H) alpha-1-antitrypsin, (I) hemopexin, and (J) alpha-1-antichymotrypsin.

Figure 7

Venn diagrams comparing ascites (asc1 and asc142) and SS proteins. (A) asc1, asc1- ConA, and asc1-WGA; (B) asc142, asc142-ConA, and asc142-WGA; (C) SS, SS-ConA, and SS-WGA; (D) asc142 and SS; (E) asc142-ConA and SS-ConA; and (F) asc142-WGA and SS-WGA.

Figure 8

(A) Clusterin sequence with N-glycosylation sites identified by bold, red, underlined type. Red arrows identify the most prevalent sites identified in serum and OC ascites samples. (B) Tandem MS/MS of two OC ascites clusterin glycopeptides with glycan and glycan site identified in each glycopeptide.

Figure 9

Hemopexin sequence, with glycan sites, number of glycopeptides identified, and % heterogeneity at each site in hemopexin present in OC ascites. (A) Hemopexin sequence. (B) Hemopexin glycan sites, composition, # peptides, and % heterogeneity in OC ascites samples #2, #4, #5, and #6.