Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pKa values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Reactive cysteine residues in proteins are typically stabilized to at least some degree in their deprotonated, thiolate form (i.e, with the pKa of the thiol lowered from an unperturbed value of about 8.7), and are important players in a variety of enzymes catalyzing redox and hydrolytic reactions through an initial nucleophilic attack on substrate [1; 2]. Unfortunately, pKa values for catalytically important cysteine residues have been determined in only a small number of cases relative to the large number of representatives for which knowledge of this value would be of biological and chemical significance. Methods for determining pKa values of cysteinyl residues in proteins typically involve the measurement of a change associated with the varying thiol/thiolate content of the protein of interest exposed to buffers over a range of pH values. Changes associated with alteration of the protonation state of a cysteine include absorbance at 240 nm, where the thiolate exhibits a modestly higher absorbance than the thiol [3; 4], NMR chemical shift changes associated with protonation/deprotonation [5; 6; 7], or changes in rates of reaction of the cysteine of interest with electrophilic chemical modifying agents wherein the more nucleophilic thiolate anion is reactive but the protonated thiol is not [8; 9; 10; 11]. In the latter case, this approach involves monitoring the progress of the chemical modification over time at each pH value; iodoacetate or its uncharged counterpart, iodoacetamide, are typically used in such studies, and progress of the reaction is measured by various methods including functional assays [11; 12; 13] or HPLC to separate unmodified from singly or doubly modified protein [14]. Particularly well studied is the thioredoxin family of proteins, where the more N-terminal of the cysteinyl residues in the CXXC motif exhibits a pKa value as low as 3.3 – 3.5 for the oxidizing protein DsbA [10; 14] or as high as 6.7 – 7.5 for the highly reducing thioredoxins [5; 7; 15; 16]. Considering methods available for pKa analyses, we sought to develop an approach to cysteinyl pKa determination based on iodoacetamide modification that takes advantage of peptide analyses by quantitative mass spectrometry. Our test protein for these studies was thioredoxin from E. coli for which chemical modification data with iodoacetamide has been previously reported [11; 14]. With the approach introduced here, proteolytic digestion and matrix assisted laser desorption ionization – time of flight (MALDI-TOF) mass spectrometry (MS) of proteins is used to monitor the extent of reaction of specific cysteinyl residues at various time points, an approach that requires very little protein and accurately maps the modification(s) to one or more specific peptides. The iodoacetamide derivative used herein can be readily synthesized in two “isotope-coded” forms, incorporating no deuterium (d0) or five deuterium atoms (d5) into the N-phenyl iodoacetamide (iodoacetanilide) reagent to distinguish the products, similar to the original “isotope coded affinity tag” (ICAT)2 technique introduced by Aebersold and colleagues [17]. In order to provide the data needed to evaluate pKa by chemical modification, a fully or partially modified d5-iodoacetanilide-labeled version of the protein is added in constant amounts to each quenched test sample taken over a time course of d0-iodoacetanilide reaction, tryptic digests are analyzed by MALDI-TOF MS for their ratio of light to heavy modified peptide to monitor reaction progress, and the modification reaction is evaluated over a range of pH values. With this approach, multiple cysteinyl sites on the same or separate peptides in a digest can be simultaneously monitored, obviating the need to study mutant proteins where one or more target cysteine residues have been replaced by serine or alanine. This mass spectrometry-based approach thus provides a readily synthesized tool and new analytical technique for conducting pKa studies of cysteine-containing proteins of interest.