Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry.
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.
Formulation and Analytical Resources, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
Da
D
Ren
Gary D
GD
Pipes
David
D
Hambly
Pavel V
PV
Bondarenko
Michael J
MJ
Treuheit
Himanshu S
HS
Gadgil
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry*; D050440:Immunoglobulin Subunits / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D007074:Immunoglobulin G; D050440:Immunoglobulin Subunits