Dimethyl isotope labeling assisted de novo peptide sequencing |
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Authors: | Marco L Hennrich Shabaz Mohammed A F Maarten Altelaar Albert J R Heck |
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Institution: | aBiomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands;bThe Netherlands Proteomics Center, Utrecht, The Netherlands;cCenter for Biomedical Genetics, Utrecht, The Netherlands |
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Abstract: | Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl
labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions.
For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific
labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous
fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal
and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due
to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins
isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues,
a previously uncharacterized avocado ascorbate peroxidase. |
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