Discrimination of Leucine and Isoleucine in Peptides Sequencing with Orbitrap Fusion Mass Spectrometer

Lebedev, A. T.; Damoc, Eugen; Makarov, Alexander; Samgina, T. Yu.

doi:10.1021/ac501200h

Cited by 69 publications

(82 citation statements)

References 32 publications

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“…6,[14][15][16] Among them, observation of their side chain fragmentation such as d-and w-ions under HE-CID conditions is the most straightforward method. With the advent of TOF-TOF instruments, access to the HE-CID analysis becomes much easier than before, which had been available only by sector instruments.…”

Section: Resultsmentioning

confidence: 99%

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

et al. 2016

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De novo sequencing is still essential in the identi cation of peptides and proteins from unexplored organisms whose sequence information is not available. One of the remaining problems in de novo sequencing is discrimination between Leu and Ile residues. e discrimination is possible based on di erences in side chain fragmentation between Leu and Ile under high-energy collision-induced dissociation (HE-CID) conditions. However, this is observed only when basic residues, such as Arg and Lys, are present near the N-or C-terminal end. It has been shown that the charge derivatization at the N-terminal end by a quarternary ammonium or phosphonium moiety facilitates the side chain fragmentation by HE-CID. However, the effective backbone fragmentation by low-energy CID (LE-CID) is o en hampered in those derivatives with a xed charge. Previously, we demonstrated that the N-terminal charge derivatization with the structures having high proton a nity induced the preferential formation of b-ions under LE-CID conditions, allowing straightforward interpretation of product ion spectra. In the present study, we further investigated whether the same derivatization approach is also e ective for discrimination between Leu and Ile under HE-CID conditions. Consequently, the side chain fragmentation of Leu and Ile residues was most e ectively enhanced by the N-terminal derivatization with 4-(guanidinomethyl)benzoic acid among the tested structures. is derivatization approach, which is compatible with both HE-and LE-CID analysis, o ers a straightforward and unambiguous de novo peptide sequencing method.

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Section: Resultsmentioning

confidence: 99%

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

et al. 2016

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“…For instance, distinguishing the isobaric isomers Leu and Ile is not possible in MS/MS data, except in some cases with specially designed fragmentation protocols that require MS instruments different to the one used in this work. 12,13 Although these 2 residues are structurally similar, antigen binding may be affected if an incorrect assignment is made, particularly within a region that makes antigen contacts or can affect the folded antibody conformation. In our work, we found that 6 of 9 library positions encoded as Leu/Ile showed a marked preference for one or the other residue following antigen binding selection.…”

Section: ---------------------------mentioning

confidence: 99%

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

Rickert¹,

Grinberg²,

Woods³

et al. 2016

mAbs

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(2016) Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies, mAbs, 8:3, 501-512, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTThe enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

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“…The corresponding losses of ethyl/methyl radical in the case of Ile, and of the isopropyl radical in the case of Leu, allow the residue to be unequivocally identified. [31] It is worth mentioning that these characteristic ions are the only fragment ions in the spectrum, while radical site migration is significantly suppressed and the result may be obtained even for the several Leu/Ile residues in a row. It is especially valuable for the amphibian peptides containing up to 8 Leu/Ile residues in the chain.…”

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confidence: 99%

LTQ Orbitrap Velos in routine de novo sequencing of non‐tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation

Samgina

Tolpina

Trebše

et al. 2015

Rapid Comm Mass Spectrometry

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Discrimination of Leucine and Isoleucine in Peptides Sequencing with Orbitrap Fusion Mass Spectrometer

Cited by 69 publications

References 32 publications

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

LTQ Orbitrap Velos in routine de novo sequencing of non‐tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation

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