Precise Leucine/Isoleucine Discrimination
Creative Biolabs has achieved the precise identification of leucine (Leu) and isoleucine (Ile) by mass spectrometry, which combines electron transfer dissociation (ETD) and high energy collision-induced dissociation (HCD) to fragment peptides.
Why is Ile/Leu Discrimination Needed?
Although Ile and leucine share exactly the same molecular weight, their different structures may dramatically influence antibody functions.
Particularly, complementarity-determining regions (CDRs) of an antibody contain at least three Ile/Leu, resulting in at least 8 expression variants which may behave significant differences in its biochemical properties, including antigen specificity, affinity, and therefore therapeutic efficacy.
How to Discriminate between Ile/Leu?
Enzyme-cleavage Specificity
Since MS/MS devices lead to more interpretable cleavage data from peptides than proteins, MS/MS proteomics procedures commonly utilize enzymes for breaking proteins down into smaller peptides. Each digestion enzyme has a propensity to cleave at specific residues.
Ile & Leu Side Chain Fragmentation by ETD & HCD
Ile & Leu side-chain fragmentation can result in MS2 fragmentation of the peptide by ETD fragmentation and MS3 of the target z ion by HCD fragmentation.
Fig. 1 ETD produces targeted z-ions. (Creative Biolabs)
Left: example spectrum for leucine, 43.05 Da mass loss of z-ion.
Right: example spectrum for isoleucine, 29.04 Da mass loss of z-ion.
The z-ions where the unknown Xle is at N-Cα bond breakage results in Leu and Ile characteristic w-ions---leucine loses a 43.05 Da diagnostic ion (C3H7), whereas isoleucine releases a 29.04 Da diagnostic ion (C2H5).
Fig. 2 Side chain fragmentation of leucine and isoleucine. (Creative Biolabs)
- The diagnostic ions can be observed for 70-80 % of all Leucine/Isoleucine residues.
- Most positions can be determined unambiguously using this method.
- The best part is an accuracy of nearly 100%.
V/J Gene Homology
Antibody sequences can be traced back to their germline gene origins and predicted Ile and Leu can be confirmed by checking the encoding codon for antibody-labeled V and J gene segments. Of note, antibodies contain CDRs that are highly prone to mutation.
At Creative Biolabs, precise discrimination of Leu/Ile can accelerate your project schedule by avoiding trying the expression of different variants, which also saves money. Please inquire us to achieve your de novo antibody sequencing.
Related Services:
- DASS Technology
- Isobaric Amino Acids Discrimination
- Manual Sequence Curation
- Intact Mass Validation
- State-of-the-Art Instruments