Direct Isoform Analysis of High-Mannose-Containing Glycoproteins by On-Line Capillary Electrophoresis Electrospray Mass Spectrometry

Yeung, Bernice; Porter, Thomas J.; Vath, James E.

doi:10.1021/ac9611172

Cited by 53 publications

(36 citation statements)

References 23 publications

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“…Therefore, microheterogeneity at single glycosylation sites introduces structural diversity resulting in various glycoforms and thus imposing considerable challenges on structural analysis. Elucidation of the glycosylation structure has either been done on the intact protein [39,169,280,281] or via enzymatic cleavage followed by analysis of the glycosylation sites and the released glycan pool [37,278]. Rizzi et al [39] separated seven isoforms of the glycoprotein antithrombin, present in widely varying abundances in human plasma, employing a PVA-coated capillary.…”

Section: Analysis Of Glycoproteins and Glycopeptidesmentioning

confidence: 99%

“…The successful separation in CZE-ESI-MS demanded the addition of 4 mol?L 21 urea to volatile acetic acid under zero-EOF conditions [39]. Yeung et al [281] also investigated glycosylation pattern on intact RNase B and human bone morphogenetic protein-2. Encountered mass differences of the glycoforms corresponding to the respective carbohydrate residues were assigned to the attached carbohydrate sequence [39,132,281].…”

Section: Analysis Of Glycoproteins and Glycopeptidesmentioning

confidence: 99%

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Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

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Section: Analysis Of Glycoproteins and Glycopeptidesmentioning

confidence: 99%

Section: Analysis Of Glycoproteins and Glycopeptidesmentioning

confidence: 99%

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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“…The presence of three CHO rhBMP-2 Nterminal species (T266, Q283, and H286) previously was documented. 18,19 It is likely that an additional pyroglutamic acid (<Q283) N-terminal species also is present, but this would be undetected with the sequencing protocol employed. Two rhBMP-4 Nterminal species were detected, differing in only a single aa residue.…”

Section: Protein Characterizationmentioning

confidence: 99%

Implantation of recombinant human bone morphogenetic proteins with biomaterial carriers: A correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model

Uludağ

D'Augusta²,

Golden³

et al. 2000

J. Biomed. Mater. Res.

181

118

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This study was carried out to determine the effect of recombinant human bone morphogenetic protein (rhBMP) pharmacokinetics (PK) on rhBMP-induced osteoinductive activity. It was our working hypothesis that the PK of a rhBMP significantly affects its osteoinductive activity. The PK of various rhBMPs (rhBMP-2, rhBMP-4, rhBMP-6, and chemically modified rhBMP-2) implanted with four biomaterial carries (Helistat, hDBM, Osteograf/N, and Dexon) was determined using (125)I-labeled proteins in the rat ectopic assay. A select combination of rhBMP and carriers then was evaluated in the rat ectopic assay for osteoinductive activity using a semi-quantitative histologic scoring system. The results indicate that initial protein retention is dependent on protein isoelectric point (pI); proteins with a higher pI yielded a higher implant retention. Subsequent PK was not strongly dependent on the pI or on the carrier. Because of the difference in early retention, the rhBMP-carrier combinations exhibited a >100-fold difference in implant-retained protein dose. When rhBMP-2 and rhBMP-4 were implanted with the carriers, more rhBMP-2 was retained in an implant, and the osteoinductive potency of rhBMP-2 typically was higher than rhBMP-4 at low implantation doses. We conclude that protein pI plays a significant role in the local retention of implanted rhBMP and that higher retention yields a higher osteoinductive activity.

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“…However, this setup enables the use of nonvolatile BGE at low electrolyte concentration. A similar approach has been shown for the separation of isoforms of ribonuclease B and recombinant human bone morphogenetic protein-2 using b-alanin as BGE in combination with on-line MS detection [170]. In a very recent study, different nonvolatile BGEs were tested in comparison to the formate-based system [182].…”

Section: Applications To Intact Proteinsmentioning

confidence: 97%

On‐line capillary electrophoresis‐mass spectrometry for the analysis of biomolecules

et al. 2004

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Mass spectrometry (MS) has become a key tool for the characterization of biologically relevant molecules in the last decade. Due to the complexity of most biological samples an upstream separation is essential. Capillary electrophoresis (CE) has gained much interest due to its high separation efficiency, speed, and often complementary selectivity to liquid chromatography. We describe the state-of-the-art of on-line CE-MS for the analysis of molecules of biological origin. The characterization of peptides, including the study of post-translational modifications, intact proteins, oligonucleotides, and related interaction studies are reviewed. Relevant publications are summarized in tables, including some important method parameters. Key applications are discussed with respect to the advantages and limitations of CE-MS. Coupling interfaces, preconcentration techniques, capillary coatings, and the different CE techniques, e.g., capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, etc. are briefly discussed against the background of their bioanalytical applications.

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Direct Isoform Analysis of High-Mannose-Containing Glycoproteins by On-Line Capillary Electrophoresis Electrospray Mass Spectrometry

Abstract: Table 3. Area Counts from A280 and SIM Analysis rhBMP-2 isoform % area from A280 % area from SIM (n ) 3) extended form 29 40 ( 2.1 mature + pyroGlu forms 71 60 ( 2.1

Cited by 53 publications

References 23 publications

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

Implantation of recombinant human bone morphogenetic proteins with biomaterial carriers: A correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model

On‐line capillary electrophoresis‐mass spectrometry for the analysis of biomolecules

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