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Electrospray‐assisted modification of proteins: a radical probe of protein structure
Author(s) -
Maleknia Simin D.,
Chance Mark R.,
Downard Kevin M.
Publication year - 1999
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19991215)13:23<2352::aid-rcm798>3.0.co;2-x
Subject(s) - chemistry , tryptophan , electrospray , radical , histidine , lysozyme , tandem mass spectrometry , cysteine , protein structure , amino acid , mass spectrometry , organic chemistry , biochemistry , chromatography , enzyme
A new approach is described to probe the structure of proteins through their reactivity with oxygen‐containing radicals. 1 Radical‐induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical‐induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher‐order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical‐induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases. Copyright © 1999 John Wiley & Sons, Ltd.