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Identification of racemization sites using deuterium labeling and tandem mass spectrometry.
Anal Chem. 2010 Aug 01; 82(15):6363-9.AC

Abstract

Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples. Immunoglobulin G subclasses IgG1, IgG2, and IgG4 samples were first stressed in protonated or deuterated buffer (pH 8 or 9) at 40 or 50 degrees C storage for days or weeks. These forced degraded samples were reduced, S-carbamidomethylated, and digested with trypsin in protonated solution, and the tryptic digests were then analyzed via liquid chromatography/mass spectrometry (LC-MS) or sequenced via liquid chromatography/tandem mass spectrometry (LC-MS/MS) to detect racemized peptides and elucidate the location of racemized amino acid residues. The methodology successfully identified several racemized amino acid residues in the constant region of the heavy chains of the three IgG subclasses. Although the IgG subclasses have very similar primary protein sequences, our results interestingly indicated different racemization rates for specific amino acid residues.

Authors+Show Affiliations

Bioproduct Research & Development, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA. huang_lihua@lilly.comNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

20669991

Citation

Huang, Lihua, et al. "Identification of Racemization Sites Using Deuterium Labeling and Tandem Mass Spectrometry." Analytical Chemistry, vol. 82, no. 15, 2010, pp. 6363-9.
Huang L, Lu X, Gough PC, et al. Identification of racemization sites using deuterium labeling and tandem mass spectrometry. Anal Chem. 2010;82(15):6363-9.
Huang, L., Lu, X., Gough, P. C., & De Felippis, M. R. (2010). Identification of racemization sites using deuterium labeling and tandem mass spectrometry. Analytical Chemistry, 82(15), 6363-9. https://doi.org/10.1021/ac101348w
Huang L, et al. Identification of Racemization Sites Using Deuterium Labeling and Tandem Mass Spectrometry. Anal Chem. 2010 Aug 1;82(15):6363-9. PubMed PMID: 20669991.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of racemization sites using deuterium labeling and tandem mass spectrometry. AU - Huang,Lihua, AU - Lu,Xiaojun, AU - Gough,P Clayton, AU - De Felippis,Michael R, PY - 2010/7/31/entrez PY - 2010/7/31/pubmed PY - 2010/12/14/medline SP - 6363 EP - 9 JF - Analytical chemistry JO - Anal Chem VL - 82 IS - 15 N2 - Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples. Immunoglobulin G subclasses IgG1, IgG2, and IgG4 samples were first stressed in protonated or deuterated buffer (pH 8 or 9) at 40 or 50 degrees C storage for days or weeks. These forced degraded samples were reduced, S-carbamidomethylated, and digested with trypsin in protonated solution, and the tryptic digests were then analyzed via liquid chromatography/mass spectrometry (LC-MS) or sequenced via liquid chromatography/tandem mass spectrometry (LC-MS/MS) to detect racemized peptides and elucidate the location of racemized amino acid residues. The methodology successfully identified several racemized amino acid residues in the constant region of the heavy chains of the three IgG subclasses. Although the IgG subclasses have very similar primary protein sequences, our results interestingly indicated different racemization rates for specific amino acid residues. SN - 1520-6882 UR - https://www.unboundmedicine.com/medline/citation/20669991/Identification_of_racemization_sites_using_deuterium_labeling_and_tandem_mass_spectrometry_ L2 - https://doi.org/10.1021/ac101348w DB - PRIME DP - Unbound Medicine ER -