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Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry.
- Source :
-
Analytical chemistry [Anal Chem] 2014 Sep 16; Vol. 86 (18), pp. 9074-81. Date of Electronic Publication: 2014 Sep 03. - Publication Year :
- 2014
-
Abstract
- Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.
- Subjects :
- Amino Acid Sequence
Antibodies, Monoclonal chemistry
Antibodies, Monoclonal metabolism
Antibodies, Monoclonal, Humanized analysis
Antibodies, Monoclonal, Humanized metabolism
Carbohydrate Sequence
Glycopeptides analysis
Molecular Sequence Data
Peptides analysis
Peptides chemistry
Protein Processing, Post-Translational
Proteomics
Trastuzumab
Trypsin metabolism
Antibodies, Monoclonal analysis
Electrophoresis, Capillary
Isotachophoresis
Tandem Mass Spectrometry
Subjects
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 86
- Issue :
- 18
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 25141158
- Full Text :
- https://doi.org/10.1021/ac502378e