Differential protein labeling based on electrochemically generated reactive intermediates.
A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as β-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments.
Westfälische Wilhelms-Universität Münster , NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany.|Westfälische Wilhelms-Universität Münster , Institut für Anorganische und Analytische Chemie, Corrensstraße 30, 48149 Münster, Germany.
Lars
L
Büter
Westfälische Wilhelms-Universität Münster , Institut für Anorganische und Analytische Chemie, Corrensstraße 30, 48149 Münster, Germany.
Helene
H
Faber
Westfälische Wilhelms-Universität Münster , NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany.|Westfälische Wilhelms-Universität Münster , Institut für Anorganische und Analytische Chemie, Corrensstraße 30, 48149 Münster, Germany.
Tina
T
Wigger
Westfälische Wilhelms-Universität Münster , Institut für Anorganische und Analytische Chemie, Corrensstraße 30, 48149 Münster, Germany.
Martin
M
Vogel
Westfälische Wilhelms-Universität Münster , NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany.|Westfälische Wilhelms-Universität Münster , Institut für Anorganische und Analytische Chemie, Corrensstraße 30, 48149 Münster, Germany.
Uwe
U
Karst
D000082:Acetaminophen / Q000737:chemistry; D000818:Animals; D016227:Benzoquinones / Q000737:chemistry; D024401:Carbonic Anhydrase I / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation; D003545:Cysteine / Q000032:analysis*; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005978:Glutathione / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling / Q000295:instrumentation / Q000379:methods*; D007782:Lactoglobulins / Q000737:chemistry; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods
D016227:Benzoquinones; D006454:Hemoglobins; D007782:Lactoglobulins; D011506:Proteins; D012709:Serum Albumin; D019800:Phenol; D000082:Acetaminophen; C004532:quinone; D024401:Carbonic Anhydrase I; D005978:Glutathione; D003545:Cysteine