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Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry.
Rapid Commun Mass Spectrom. 2005; 19(20):2965-73.RC

Abstract

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.

Authors+Show Affiliations

Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16178042

Citation

Kleinova, Martina, et al. "Characterization of Cysteinylation of Pharmaceutical-grade Human Serum Albumin By Electrospray Ionization Mass Spectrometry and Low-energy Collision-induced Dissociation Tandem Mass Spectrometry." Rapid Communications in Mass Spectrometry : RCM, vol. 19, no. 20, 2005, pp. 2965-73.
Kleinova M, Belgacem O, Pock K, et al. Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(20):2965-73.
Kleinova, M., Belgacem, O., Pock, K., Rizzi, A., Buchacher, A., & Allmaier, G. (2005). Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry. Rapid Communications in Mass Spectrometry : RCM, 19(20), 2965-73.
Kleinova M, et al. Characterization of Cysteinylation of Pharmaceutical-grade Human Serum Albumin By Electrospray Ionization Mass Spectrometry and Low-energy Collision-induced Dissociation Tandem Mass Spectrometry. Rapid Commun Mass Spectrom. 2005;19(20):2965-73. PubMed PMID: 16178042.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry. AU - Kleinova,Martina, AU - Belgacem,Omar, AU - Pock,Katharina, AU - Rizzi,Andreas, AU - Buchacher,Andrea, AU - Allmaier,Günter, PY - 2005/9/24/pubmed PY - 2005/11/16/medline PY - 2005/9/24/entrez SP - 2965 EP - 73 JF - Rapid communications in mass spectrometry : RCM JO - Rapid Commun Mass Spectrom VL - 19 IS - 20 N2 - Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation. SN - 0951-4198 UR - https://www.unboundmedicine.com/medline/citation/16178042/Characterization_of_cysteinylation_of_pharmaceutical_grade_human_serum_albumin_by_electrospray_ionization_mass_spectrometry_and_low_energy_collision_induced_dissociation_tandem_mass_spectrometry_ L2 - https://doi.org/10.1002/rcm.2154 DB - PRIME DP - Unbound Medicine ER -