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  • P-II-0400

Evaluation of novel chemical derivatization methods for N-Terminal sequencing of proteins

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New Technology: Sample Preparation

Poster

Evaluation of novel chemical derivatization methods for N-Terminal sequencing of proteins

Thema

  • New Technology: Sample Preparation

Mitwirkende

Peter Gehrig (Zurich / CH), Simone Wüthrich (Zurich / CH), Ralph Schlapbach (Zurich / CH), Paolo Nanni (Zurich / CH)

Abstract

Introduction.

N-terminal protein sequencing permits reliable identification of real N-termini of mature proteins, and it provides essential insights into protease substrates and specificity. A critical step in MS-based N-terminomics workflows is the modification of primary amines present at the protein N-terminus and in lysine residues. Reductive methylation is such a strategy, which has been widely used for N-terminal protein sequencing. Chemically, two methyl groups are added to primary amines in a mixture of formaldehyde and sodium cyanoborohydride. In this study, new methods for N-terminal modification of proteins were introduced and applied to commercially available proteins. For the dimethylation reaction, we replaced the commonly applied, but highly poisonous reducing reagent sodium cyanoborohydride by pyridine borane, which showed a similar reactivity. Furthermore, we modified free amino groups with nicotinic acid N-hydroxysuccinimide (NHS) ester as an alternative to the dimethylation reaction. Finally, we evaluated the one-step reaction with 2-pyridinecarboxaldehyde (2PCA), which is specific for protein N-termini due to a unique cyclization reaction involving the second amino acid.

Methods.

Selected proteins (BSA, myoglobin, α-casein, α-lactalbumin, α-casein, ß-galactosidase) were modified with a mixture of formaldehyde and pyridine borane, nicotinic acid NHS ester, or 2PCA. The reagents were quenched and removed, and the labeled proteins were cleaved with trypsin or chymotrypsin using the SP3 approach. The resulting peptides were subjected to LC-MS and MALDI-TOF analysis.

Results and Conclusions.

Important criteria for chemical derivatization methods are complete reactions and the absence of undesired side reactions. In addition, chemical modifications of peptides should have a favorable influence on MS signal intensities and on the fragmentation spectra.

The results for the dimethylation reaction showed that pyridine borane promotes dimethylation at levels comparable to those recommended for the standard reducing agent sodium cyanoborohydride. Nearly complete dimethylation of N-termini and lysines was achieved, and we recommend replacement of the highly toxic cyanoborohydride by pyridine borane for this application.

Nearly quantitative reactions and only minor side reactions were observed for the nicotinic acid NHS ester modification of proteins. Interestingly, tandem mass spectra acquired from peptides carrying this modification gave higher database search scores due to more intense b ions including b1 ions, which are not detectable otherwise.

Our preliminary results confirm that the reaction with 2PCA is largely specific for N-terminal amines. Further optimization is required to increase the yield beyond approximately 50%. 2PCA labeling is complementary to most N-terminal labeling methods since lysine residues remain unmodified and are available for cleavage by proteases.

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