Specific cleavage of blotted proteins at cysteine residues after cyanylation: Analysis of products by MALDI-TOF

Abstract

Publisher Summary This chapter describes efficient procedures for cleaving proteins bound to polyvinylidene difluoride (PVDF) membranes. The reagent 2-nitro-5-thiocyanobenzoic acid (NTCB) is ideal for cleaving proteins at fully reduced Cys residues under relatively mild conditions. The cleavage is efficient and offers high potential for distinguishing cysteine residues from cystine residues in proteins where both are present and for assigning disulfide bonds. The chapter describes a procedure for cleaving proteins with NTCB after they have been separated by sodium dodecyl sulfate (SDS) gel electrophoresis and electro-blotted to PVDF membranes. Cyanylation occurs best at pH 8 with a 4-fold excess of NTCB over SH groups, whereas cleavage occurs best at pH 9. However, after cyanylation, there is always equilibrium between cleavage and β-elimination. The resulting fragments are analyzed by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, an analytical method that is powerful for identifying resulting fragments simply by their mass. Most of the expected fragments are found for complete cleavage. Analyzing the products by MALDI-TOF gives a clear idea of the extent of the side reactions.