Full Antibody Primary Structure and Microvariant Characterization
in a Single Injection Using Transient Isotachophoresis and Sheathless
Capillary Electrophoresis–Tandem Mass Spectrometry
- Publication date
- Publisher
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