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Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease.

Benicky J, Sanda M, Pompach P, Wu J, Goldman R - Anal. Chem. (2014)

Bottom Line: We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples.Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX.The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University , LCCC Room S183, 3970 Reservoir Rd NW, Washington, D.C., 20057, United States.

ABSTRACT
Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

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Core fucosylation inpooled samples of healthy controls (H), cirrhosis(CIR), and HCC patients. Core fucosylation of (A) hemopexin and (B)CFH was analyzed following endoglycosidase F2/F3 treatment. Relativeabundance of each fucosylated glycoform, quantified as area of precursorion XIC peak, is presented as a percent of its nonfucosylated counterpart.The position of the glycosylation site in the protein sequence isshown below the corresponding group of bars representing three patientgroups. Results are shown as mean ± SD; ∗, P < 0.05 vs H; ND, nondetectable.
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fig3: Core fucosylation inpooled samples of healthy controls (H), cirrhosis(CIR), and HCC patients. Core fucosylation of (A) hemopexin and (B)CFH was analyzed following endoglycosidase F2/F3 treatment. Relativeabundance of each fucosylated glycoform, quantified as area of precursorion XIC peak, is presented as a percent of its nonfucosylated counterpart.The position of the glycosylation site in the protein sequence isshown below the corresponding group of bars representing three patientgroups. Results are shown as mean ± SD; ∗, P < 0.05 vs H; ND, nondetectable.

Mentions: Thepreceding analysis does not allow unequivocal differentiation of coreα(1–6) linkage from outer arm fucosylation; these modificationsare carried out by a different set of enzymes and are important todistinguish in the liver disease context.11,19,52 To quantify core fucosylation, we treatedglycopeptides with a combination of endoglycosidase F2 and F3. Thiscleaves complex bi- and triantennary glycans leaving the innermostN-linked GlcNAc with or without core fucose attached to the peptide. We have utilized both enzymes in excess and did not observe any residualuncleaved glycopeptides after overnight digest. The proportion ofcore fucosylated peptides, expressed as a percent of the nonfucosylatedform, is presented in Figure 3. In the caseof HPX, core fucose was identified on all three singly glycosylatedpeptides. The percentage of core fucose in healthy subjects was below5% on all sites; a tendency toward an increased ratio of fucosylatedstructures was observed in liver disease, but this increase was minor(Figure 3A). In contrast, core fucosylationof CFH was clearly elevated in the liver disease groups at N217 andN1029 positions (Figure 3B), but a differencebetween cirrhosis and HCC groups was not observed. We did not detectany core fucosylation at N882 and N911 sites of CFH although the nonfucosylatedglycopeptides were readily detected. This indicates that the changesin overall fucosylation at these sites (Figures 1, 2, and 4) are associatedwith alterations in outer arm fucosylation.


Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease.

Benicky J, Sanda M, Pompach P, Wu J, Goldman R - Anal. Chem. (2014)

Core fucosylation inpooled samples of healthy controls (H), cirrhosis(CIR), and HCC patients. Core fucosylation of (A) hemopexin and (B)CFH was analyzed following endoglycosidase F2/F3 treatment. Relativeabundance of each fucosylated glycoform, quantified as area of precursorion XIC peak, is presented as a percent of its nonfucosylated counterpart.The position of the glycosylation site in the protein sequence isshown below the corresponding group of bars representing three patientgroups. Results are shown as mean ± SD; ∗, P < 0.05 vs H; ND, nondetectable.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4222631&req=5

fig3: Core fucosylation inpooled samples of healthy controls (H), cirrhosis(CIR), and HCC patients. Core fucosylation of (A) hemopexin and (B)CFH was analyzed following endoglycosidase F2/F3 treatment. Relativeabundance of each fucosylated glycoform, quantified as area of precursorion XIC peak, is presented as a percent of its nonfucosylated counterpart.The position of the glycosylation site in the protein sequence isshown below the corresponding group of bars representing three patientgroups. Results are shown as mean ± SD; ∗, P < 0.05 vs H; ND, nondetectable.
Mentions: Thepreceding analysis does not allow unequivocal differentiation of coreα(1–6) linkage from outer arm fucosylation; these modificationsare carried out by a different set of enzymes and are important todistinguish in the liver disease context.11,19,52 To quantify core fucosylation, we treatedglycopeptides with a combination of endoglycosidase F2 and F3. Thiscleaves complex bi- and triantennary glycans leaving the innermostN-linked GlcNAc with or without core fucose attached to the peptide. We have utilized both enzymes in excess and did not observe any residualuncleaved glycopeptides after overnight digest. The proportion ofcore fucosylated peptides, expressed as a percent of the nonfucosylatedform, is presented in Figure 3. In the caseof HPX, core fucose was identified on all three singly glycosylatedpeptides. The percentage of core fucose in healthy subjects was below5% on all sites; a tendency toward an increased ratio of fucosylatedstructures was observed in liver disease, but this increase was minor(Figure 3A). In contrast, core fucosylationof CFH was clearly elevated in the liver disease groups at N217 andN1029 positions (Figure 3B), but a differencebetween cirrhosis and HCC groups was not observed. We did not detectany core fucosylation at N882 and N911 sites of CFH although the nonfucosylatedglycopeptides were readily detected. This indicates that the changesin overall fucosylation at these sites (Figures 1, 2, and 4) are associatedwith alterations in outer arm fucosylation.

Bottom Line: We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples.Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX.The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University , LCCC Room S183, 3970 Reservoir Rd NW, Washington, D.C., 20057, United States.

ABSTRACT
Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

Show MeSH
Related in: MedlinePlus