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  • Title: Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry.
    Author: Ren D, Pipes GD, Hambly D, Bondarenko PV, Treuheit MJ, Gadgil HS.
    Journal: Anal Biochem; 2009 Jan 01; 384(1):42-8. PubMed ID: 18834850.
    Abstract:
    An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues.
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