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-MS) and ETD (electron transfer dissociation)-HCD MS analysis 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.

The Role of Electron Transfer Dissociation in Modern Proteomics

“… 288 Lebedev et al and Xiao et al both recently described methods to perform HCD on z • -type generated from ETD to create diagnostic w-ions for distinguishing leucine and isoleucine residues in peptides. 289 , 290 Lebedev and co-workers further extended this study to EThcD methods for more globally applicable leucine/isoleucine discrimination, 291 although they also described limitations and considerations when using these approaches. 292 Lyon et al recently showed that ETD reactions can identify peptide isomers through a strategy that leverages hemolytic cleavage of carbon–iodine bonds that drives radical directed dissociation upon supplemental activation.…”

PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor

“…An MS 3 method 49 was implemented to assign the unknown residue at position 2 because MS/MS fragmentation alone cannot be used to distinguish between the isobaric residues leucine and isoleucine. This approach takes advantage of the differing HCD fragmentation patterns of Ile and Leu immonium ions.…”

De Novo Sequencing of Antibody Light Chain Proteoforms from Patients with Multiple Myeloma

“…For isoleucine/leucine discrimination, trypsin and chymotrypsin LC digests were mixed (1:1 ratio) and ions corresponding to peptides containing one or more leucine or isoleucine were added to an inclusion list to be fragmented by EThcD (MS 2 ) or HCD (MS 4 ) as previously described. 37,38 De novo peptide sequencing and concatenation PEAKS Studio X was used for peptide de novo sequencing. The data were refined using precursor mass correction only and the chimera scan option activated.…”