title: Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis Using an Orbitrap Fusion Mass Spectrometer.
abstract: Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their molecular masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, especially in CDRs (complementarity determining regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, we describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissociation) multistage mass spectrometric analysis (HCD-MSn) and ETD (electron transfer dissociation)-HCD MS3analysis using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides. The merits and limitations of this Leu/Ile discrimination approach are evaluated. Using the new approach, along with proposed decision-making guidelines we unambiguously identified every Leu/Ile residue in peptides containing up to five Leu/Ile residues and molecular masses up to 3000 Da. In addition, we have demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly determined using this approach. Our results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry. In principle, this integrated, online LC-MS approach for Leu/Ile assignment can be applied to de novo sequencing of any protein or peptide.
authors.affiliation: Analytical Biochemistry, Department of Cell and Protein Sciences, Biogen , 250 Binney Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Analytical Biochemistry, Department of Cell and Protein Sciences, Biogen , 250 Binney Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Malgorzata M
authors.initials: MM
authors.lastname: Vecchi
authors.affiliation: Analytical Biochemistry, Department of Cell and Protein Sciences, Biogen , 250 Binney Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Dingyi
authors.initials: D
authors.lastname: Wen
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D007532:Isoleucine / Q000032:analysis*; D007930:Leucine / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D007532:Isoleucine; D007930:Leucine