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.
Ren
Da
D
Formulation and Analytical Resources, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
Pipes
Gary D
GD
Hambly
David
D
Bondarenko
Pavel V
PV
Treuheit
Michael J
MJ
Gadgil
Himanshu S
HS
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