Strategy for selective chemical cross-linking of tyrosine and lysine residues.
Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.
Leavell
Michael D
MD
Sandia National Laboratories, Livermore, California 94551-0969, USA.
Novak
Petr
P
Behrens
Christopher R
CR
Schoeniger
Joseph S
JS
Kruppa
Gary H
GH
D000803:Angiotensin I / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014443:Tyrosine / Q000737:chemistry*
D003432:Cross-Linking Reagents; D007328:Insulin; D014443:Tyrosine; D000803:Angiotensin I; D008239:Lysine