Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing

Clerc, François; Monegier, B.; Faucher, Didier; Cuine, F.; Pourcet, C.; Holt, John Clifford; Tang, Sheng-Yuh; Dorsselaer, Alain Van; Becquart, Jérôme; Vuilhorgne, Marc

doi:10.1016/0378-4347(94)00184-7

Cited by 6 publications

(5 citation statements)

References 25 publications

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“…These results are comparable to previous reports on the proteolytic mapping of HSA and of HSA adducts using mass spectral characterization by fast atom bombardment ionization ( − ) and by electrospray ionization ( , ).…”

Section: Resultssupporting

confidence: 89%

Structural Determination of the Conjugate of Human Serum Albumin with a Mitomycin C Derivative, KW-2149, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Yasuzawa

Tomer²

1997

Bioconjugate Chem.

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A new mitomycin C derivative, KW-2149, is known to form a covalent conjugate with human serum albumin (HSA). This conjugate exhibits 1/20 of the anticellular activity of unconjugated KW-2149. Structural studies of this conjugate were carried out using a combination of enzymatic digestion, high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The tryptic peptide T5 (residues 21-41) was the only peptide found to be modified by KW-2149 moieties, the [(gamma-L-glutamylamino)ethyl]thio group or the (2-aminoethyl)thio group, through a disulfide bond. Although the latter peptide lost its mitomycin C moiety in the course of tryptic digestion, these data strongly suggest that KW-2149 was bound to Cys-34, the only free cysteine on HSA.

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Section: Resultssupporting

confidence: 89%

Structural Determination of the Conjugate of Human Serum Albumin with a Mitomycin C Derivative, KW-2149, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Yasuzawa

Tomer²

1997

Bioconjugate Chem.

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“…The bottom trace on Figure 5A represents the Maximum Entropy data analysis after the subtraction of the overlap region to eliminate most of the 66,550 Da component. These findings are consistent with previous observations for HSA [22,23].…”

Section: Figuresupporting

confidence: 95%

Separation of human serum albumin components by RP-HPLC and CZE and their characterization by ESI-MS

et al. 1999

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SummaryHuman serum albumin (HSA) is one of the most abundant human proteins and has been shown to be heterogeneous. A RP-HPLC method has been developed to separate HSA components in commercially available preparations. Separations were carried out on Aquapore RP-300, C8 columns using gradient elution with a combination of acetonitrile/water mobile phases containing 0.05 % trifluoroacetic acid as ion-pairing agent. Optimum resolution was attained on narrow-bore columns using a stepwise, linear gradient that incorporated a shallow intermediate step of 0.20 %/min in Mobile Phase B. Under similar elution conditions, separations carried out on standard-size columns showed the expected decrease in resolution due to increased peak widths. A comparative analysis of three commercial products highlighted qualitative and quantitative differences. Capillary zone electrophoresis was used for the analysis of collected RP-HPLC fractions. Results indicated that while the HPLC separation was incomplete, one of the major HPLC peaks was primarily composed of one of the three main components typically separated by CZE. ESI-MS was used to characterize the two major RP-HPLC fractions and also showed that the HPLC separation was incomplete. The MaxEnt transform of the HPLC peaks was consistent with components all being HSA and closely related derivatives.

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“…The relative molecular mass ( M r ) of HSA, calculated from the cDNA sequence of the protein, is 66 438 ± 3 (the uncertainty is the standard deviation of the calculated relative molecular mass resulting from the uncertainty in the value of the isotopic abundance of each element 15 ). By combining Edman degradation, proteolytic cleavage, and ESI-MS, Clerc et al . verified 99% of the amino acid sequence of a recombinant HSA derived from the cDNA sequence, which had an observed M r of 66 460, slightly higher than expected, which the authors attribute to salt impurities.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Glycation of Albumin in Freeze-Dried and Frozen Human Serum

Bunk

1997

Anal. Chem.

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Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.

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Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing

Cited by 6 publications

References 25 publications

Structural Determination of the Conjugate of Human Serum Albumin with a Mitomycin C Derivative, KW-2149, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Structural Determination of the Conjugate of Human Serum Albumin with a Mitomycin C Derivative, KW-2149, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Separation of human serum albumin components by RP-HPLC and CZE and their characterization by ESI-MS

Characterization of the Glycation of Albumin in Freeze-Dried and Frozen Human Serum

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