Discrimination of Isoleucine and Leucine by Dimethylation-Assisted MS3

Abstract

Protein sequencing by mass spectrometry has transformed the field of biopharmaceutical analysis, but a missing part in the analytical toolkit is the ability to distinguish between the isomeric residues isoleucine and leucine because it is a requisite for efficient analysis of the primary structure of proteins. To address this need, we have developed a novel mass spectrometric method that combines reductive dimethylation and MS3 fragmentation with LCMS peptide mapping. The dimethylation of peptide N-termini leads to intense a1-ions upon collision-induced fragmentation, and further fragmentation of the isoleucine/leucine a1-ion leads to informative spectra with fragments that can discriminate between the two isomers. The methodology of a1-directed MS3 was applied to two antibodies in combination with the proteases trypsin, thermolysin, chymotrypsin, and pepsin to generate peptides exposing N-terminal I/L residues.

Ion Activation Methods for Peptides and Proteins

“…Alternatively, ETD may be used to generate z ions via backbone cleavage N-terminal to Ile/Leu residues. Subsequent HCD produces side-chain losses of either 43 or 29 Da that identify the residue as either leucine or isoleucine, respectively. ,, Although these multistage strategies have limited utility for some peptides based on their sizes or charge states, these methods are complementary to one another and have been demonstrated on an Orbitrap platform on an LC time scale. , Multienzyme digestion and the described LC-MS n strategies have been used to successfully distinguish Ile and Leu residues in the CDR regions of mAb1, mAb2, and the Waters mAb standard. , Similarly, multistage HCD of dimethylated peptides also resulted in diagnostic a 1 product ions for Ile/Leu, resulting in the identification of 71% to 94% of the Ile/Leu residues on the heavy and light chains of dimethylated SILuLite and Trastuzumab mAbs, respectively . When the differentiation of isomeric peptides was allowed, these new MS/MS methods advanced the feasibility of de novo sequencing through mass spectrometry.…”

“…91,92 Multienzyme digestion and the described LC-MS n strategies have been used to successfully distinguish Ile and Leu residues in the CDR regions of mAb1, mAb2, and the Waters mAb standard. 91,92 Similarly, multistage HCD of dimethylated peptides also resulted in diagnostic a 1 product ions for Ile/Leu, 95 resulting in the identification of 71% to 94% of the Ile/Leu residues on the heavy and light chains of dimethylated SILuLite and Trastuzumab mAbs, respectively. 95 When the differentiation of isomeric peptides was allowed, these new MS/MS methods advanced the feasibility of de novo sequencing through mass spectrometry.…”

“…91,92 Similarly, multistage HCD of dimethylated peptides also resulted in diagnostic a 1 product ions for Ile/Leu, 95 resulting in the identification of 71% to 94% of the Ile/Leu residues on the heavy and light chains of dimethylated SILuLite and Trastuzumab mAbs, respectively. 95 When the differentiation of isomeric peptides was allowed, these new MS/MS methods advanced the feasibility of de novo sequencing through mass spectrometry. Lacking defined termini, nonlinear peptides like cyclotides and stapled peptides present a unique challenge to tandem mass spectrometry for characterizing primary structure.…”

Characterization of HLA-A*02:01 MHC Immunopeptide Antigens Enhanced by Ultraviolet Photodissociation Mass Spectrometry

“…Some methods have addressed this limitation; however, they require either more complex MS n staging or derivatization of the peptides. 28,31 Additionally, while d and w ions were first observed with high energy (keV) CAD, 32 the production of d and/or w ions via ultraviolet photodissociation (UVPD) or EThcD is more readily accomplished using modern highperformance mass spectrometers.…”

“…CAD has also been implemented to differentiate leucine and isoleucine, typically via an MS 3 strategy, in which the 86 Da immonium ion, specific to either leucine or isoleucine, is analyzed. − This MS 3 method is not successful in characterizing peptides for which multiple leucine or isoleucine residues are present, as is the case for many immunopeptides. Some methods have addressed this limitation; however, they require either more complex MS n staging or derivatization of the peptides. , Additionally, while d and w ions were first observed with high energy (keV) CAD, the production of d and/or w ions via ultraviolet photodissociation (UVPD) or EThcD is more readily accomplished using modern high-performance mass spectrometers.…”