Study of primary amines for nucleophilic cleavage of cyanylated cystinyl proteins in disulfide mass mapping methodology Jose ´-Luis Gallegos-Pe ´rez a , Laura Rangel-Ordo ´n ˜ez b , Stephen Robert Bowman b , Charles O. Ngowe b , J. Throck Watson a,b, * a Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA b Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA Received 26 July 2005 Available online 25 August 2005 Abstract In a study of primary (methyl to butyl) amines as nucleophiles for cyano-induced cleavage of cysteinyl proteins, methylamine was found to be superior to ammonia for cyanylation (CN)-based disulfide mass mapping methodology. Reaction conditions such as nucle- ophile concentration, temperature, and reaction time were systematically studied using ribonuclease A as a model protein. The CN-in- duced cleavage products were monitored using reverse-phase chromatography and matrix-assisted laser desorption ionization mass spectrometry. Results showed that low temperature, short reaction time, and high nucleophile concentration optimize the cleavage reac- tion and minimize side reactions. These conditions shorten the analysis time and substantially improve the yield of cleavage products. Further, the concurrent use of homologous nucleophiles (e.g., ammonia and methylamine) facilitates recognition and identification of cleavage products. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Disulfide bonds; Partial reduction; Cyanylation; Protein cleavage; Chemical cleavage; Protein structure Determination of the three-dimensional structure of proteins is important for elucidating their biological activ- ity and function. Disulfide bonds are important for the sta- bilization of the native structures of cystinyl proteins, and in some cases they modulate changes in the bioactivity of the protein [1,2]. Improved methodology for determining the disulfide structure of cystinyl proteins will facilitate development of disulfide proteomics [1,3]. The first studies on the disulfide linkages in insulin and ribonuclease [4–6] established the basic and conventional methodology for determining the disulfide structure in a cystinyl protein, relying on a combination of proteolytic digestion, Edman sequencing, and mass mapping [7,8]. The drawbacks of the conventional approach to disulfide mapping include the inability to analyze proteins with adja- cent cysteines and a risk of forming artifacts due to disul- fide scrambling during any alkaline proteolysis [9–11]. The concept and strategy of partial reduction of a multi- cystinyl protein established a new methodological ap- proach to analyzing cysteine-rich peptides and proteins [12]; progressive stages of partial reduction can be marked chemically with different alkylating reagents [13], including those that form charged derivatives to facilitate subsequent analysis by MS [11], and in many cases sequencing of the chemically labeled partially reduced isoforms provided information relating to the positions of cysteines involved in particular disulfide bonds [11,13]. During the late 1990s, Wu and Watson [8] established a cyanylation (CN) 1 -based chemical method for disulfide mass mapping to overcome many of the shortcomings of the conventional proteolytic approach. The method is based on five main steps: (1) the cystinyl protein is partially 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.08.003 * Corresponding author. Fax: +1 517 353 9334. E-mail address: watsonj@msu.edu (J.T. Watson). 1 Abbreviations used: CN, cyanylation; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; Nu, nucleophile. www.elsevier.com/locate/yabio Analytical Biochemistry 346 (2005) 311–319 ANALYTICAL BIOCHEMISTRY