Journal: Journal of glycomics & lipidomics

Article Title: Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides

doi: 10.4172/2153-0637.1000129

Figure Lengend Snippet: Isolation of hFSH glycoform preparations, FSH24 and FSH21. A. Sephacryl G-100 chromatography of 1502 mg soluble human pituitary glycoprotein fraction on a 5 × 200 cm column equilibrated in 0.126 M ammonium bicarbonate at a flow rate of 150 mL/h. FSH was found in fractions A-C by RIA (dotted squares). B. A 111 mg sample of G-100A was chromatographed on a 5 × 15 cm phenyl-Sepharose column. The chromatogram was developed with: 1 M ammonium sulfate in 0.05 M sodium phosphate buffer, pH 7.0, followed by 0.2 M ammonium sulfate in 0.05 M sodium phosphate buffer, pH 7.0, 0.05 M sodium phosphate buffer, pH 7.0, and 40% ethanol/water. FSH emerged in fractions A, B, and D (inset bar graph). C. A 155 mg sample of G-100B was applied to the phenyl-Sepharose column and the chromatogram developed as in Fig. 1B. FSH largely emerged in Fraction B1 (inset bar graph). D. A 349 mg sample of G100-C was applied to the phenyl-Sepharose column. FSH was recovered in fractions A2, B1 and B2 (inset bar graph). E. FSH in fraction B1 from panel C was immunopurified using anti-hFSH monoclonal antibody 4882. F. A 51 mg sample of fractions A2, B1, and B2 from panel D was was applied to a 2.5 × 200 cm Sephacryl S-100 and the chromatogram developed with with 0.126 M ammonium bicarbonate buffer at a flow rate of 45 mL/h. FSH was found in fractions B and C (upper inset). G-I. Triple-Superdex 75 gel filtration of purified hFSH. Three 1 × 30 cm Superdex 75 columns were connected in series and equilibrated with 0.2 M ammonium bicarbonate containing 20% acetonitrile. The chromatogram was developed with the same buffer at a flow rate of 0.3 mL/min. Individual fractions were collected and samples of each fraction associated with the FSH peak analyzed by FSHβ (panel G inset) or FSHβ and FSHα-specific Western blots (panels H and I, insets).

Article Snippet: Highly purified urinary hFSH was obtained from ProSpec (East Brunswick, NJ).

Techniques: Isolation, Chromatography, Filtration, Purification, Western Blot

Journal: Journal of glycomics & lipidomics

Article Title: Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides

doi: 10.4172/2153-0637.1000129

Figure Lengend Snippet: Negative mode nano-ESI mass spectrometry of oligosaccharides released from reduced, carboxymethylated hFSH samples by peptide-N-glycanase F digestion. Structures of the more abundant glycans are shown for each preparation along with the monoisotopic m/z value. Because the 13C ion is often more abundant than the former, peaks may be labeled with m/z values that do not match values found in Table 1. A. Glycans from pituitary hFSH preparation AFP7298A. B. Glycans from purified urinary hFSH. C. Glycans from recombinant GH3-hFSH. D. Glycans from hFSH24 glycoform preparation. E. Glycans from hFSH21 glycoform preparation. F. Glycans from hypo-glycosylated hFSH21/18 preparation isolated from highly purified hLH preparations. Note the expanded scale indicated by the dashed line.

Article Snippet: Highly purified urinary hFSH was obtained from ProSpec (East Brunswick, NJ).

Techniques: Mass Spectrometry, Labeling, Purification, Recombinant, Isolation

Journal: Journal of glycomics & lipidomics

Article Title: Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides

doi: 10.4172/2153-0637.1000129

Figure Lengend Snippet: Neuraminidase-digested oligosaccharides analyzed by nano-ESI mass spectrometry. Aliquots of samples analyzed in Fig. 2 were digested with Arthrobacter ureafaciens sialidase prior to analysis. Structures of the more abundant neutral core glycans (Table 1) are shown in each panel. A. Desialylated glycans from purified pituitary hFSH. B. Desialylated glycans from purified urinary hFSH. C. Desialylated from recombinant GH3-hFSH. D. Desialylated glycans from hFSH24. E. Desialylated glycans from hFSH21. F. Desialylated glycans from hypo glycosylated hFSH21/18. Note the expanded scale indicated by the dashed line.

Article Snippet: Highly purified urinary hFSH was obtained from ProSpec (East Brunswick, NJ).

Techniques: Mass Spectrometry, Purification, Recombinant

Journal: Journal of glycomics & lipidomics

Article Title: Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides

doi: 10.4172/2153-0637.1000129

Figure Lengend Snippet: Comparison of glycan abundance by neutral core structure. The abundances of all variants sharing a common core were summed and plotted. Comparisons were made between highly purified pituitary hFSH glycans and those of the other five preparations analyzed in this study. A. Purified pituitary hFSH glycans, showing the glycan families accounting for at least 4% of the total. B. Comparison of urinary with pituitary hFSH glycans. The structures in this and subsequent panels represent urinary glycans, as pituitary glycans were shown in panel A, above. C. Comparison of recombinant GH3-hFSH with pituitary hFSH glycans. D. Comparison of hFSH24 with pituitary hFSH glycans. E. Comparison of hFSH21 with pituitary hFSH glycans. F. Comparison of hypo-glycosylated hFSH21/18 with pituitary hFSH glycans. The solid bars represent the hFSH glycan families while the gray bars represent the other hFSH preparation glycans.

Article Snippet: Highly purified urinary hFSH was obtained from ProSpec (East Brunswick, NJ).

Techniques: Comparison, Glycoproteomics, Purification, Recombinant

Journal: Journal of glycomics & lipidomics

Article Title: Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides

doi: 10.4172/2153-0637.1000129

Figure Lengend Snippet: FSH characterization by size exclusion chromatography and Western blot analysis. Samples of hFSH24, hFSH24/21, and hFSH21 were applied to a 1 × 30 cm Superdex 75 column and the chromatogram developed with 20% acetonitrile in 0.2 M ammonium bicarbonate at a flow rate of 0.4 ml/min. Retention times (min) for each FSH peak are indicated. A. Fully-glycosylated hFSH24 from Superdex 75 fraction A (Fig. 1G). Inset: Lane 1, fraction A; lane 2, fraction A’; lane 3, fraction B; lane 4, fraction B’; lane 5, fraction C; lane 6, fraction C’; lane 7, purified pituitary hFSH24/21. B. Mixture of both hFSH glycoforms. C. Hypo-glycosylated hFSH21 from fraction C, Fig. G.

Article Snippet: Highly purified urinary hFSH was obtained from ProSpec (East Brunswick, NJ).

Techniques: Size-exclusion Chromatography, Western Blot, Purification