Identification of Racemization Sites Using Deuterium Labeling and Tandem Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 82; no. 15; pp. 6363 - 6369
• Main Authors: Huang, Lihua, Lu, Xiaojun, Gough, P. Clayton, De Felippis, Michael R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-08-2010
• Subjects: Amino Acid Sequence; Amino acids; Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid; Deuterium - chemistry; Deuterium Exchange Measurement; Exact sciences and technology; Hydrogen-Ion Concentration; Immunoglobulin G - chemistry; Immunoglobulin G - metabolism; Immunoglobulins; Isomerism; Isotopes; Mass spectrometry; Molecular Sequence Data; Other chromatographic methods; Peptides; Tandem Mass Spectrometry - methods; Temperature; Trypsin - metabolism; Deuterium; Immunoglobulins; Collisional activation; Peptides; Coupled method; Sample; IgG; Racemization; Liquid chromatography; Identification; Chemical degradation; Protein; Storage; Residue; Aminoacid; Mass spectrometry MS/MS; pH; Dissociation; Mass spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac101348w

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 °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.