Mass Tagging Approach for Mitochondrial Thiol Proteins

Abstract

A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d(0) and IBTP-d(15). The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d(0)/d(15)-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications.

Protein Adducts of Aldehydic Lipid Peroxidation Products

“…A scenario is emerging in which the dielectric constant, surface accessibility and the modulation of the nucleophilicity and basicity of a certain residue by its microenvironment may govern the propensity of a distinct site to modifications by electrophiles (Roe et al, 2007; Sayre et al, 2006). In this context, various alkylating reagents have been employed to determine preferred target sites of modifications (Marley et al, 2005; Shin et al, 2007; Wong and Liebler, 2008). We explored the use of a sterically demanding butyltriphenylphosphonium group as a chemical tool for the identification of mitochondrial protein thiols that exhibit a large degree of surface accessibility (Marley et al, 2005).…”

“…In this context, various alkylating reagents have been employed to determine preferred target sites of modifications (Marley et al, 2005; Shin et al, 2007; Wong and Liebler, 2008). We explored the use of a sterically demanding butyltriphenylphosphonium group as a chemical tool for the identification of mitochondrial protein thiols that exhibit a large degree of surface accessibility (Marley et al, 2005). In this study, Cys-385 of aconitase was readily alkylated by (4-iodo)butyltriphenylphosphonium iodide (IBTP).…”

Tandem Mass Spectrometric Characterization of Thiol Peptides Modified by the Chemoselective Cationic Sulfhydryl Reagent (4-Iodobutyl)Triphenylphosphonium—

“…The alkylation of mitochondrial proteins with IBTP was described previously [11]. Briefly, rat cardiac mitochondria were isolated according to Suh, et.…”

“…As a lipophilic and cationic reagent, IBTP is able to penetrate the lipid bilayers and accumulated inside the mitochondria due to the large membrane potential present across the inner mitochondrial membrane. Our group has explored the use of stable isotopically-coded IBTP probes in gel-free proteomics approaches to obtain site-specific information on reactive thiol groups in mitochondrial proteins [11]. During the course of these studies we observed that the introduction of the butyltriphenylphosphonium (BTP)-moiety affected the collision-induced dissociation (CID) behaviors of BTP-modified peptides which warranted further investigations.…”

Detection of carbonyl‐modified proteins in interfibrillar rat mitochondria using N′‐aminooxymethylcarbonylhydrazino‐D‐biotin as an aldehyde/keto‐reactive probe in combination with Western blot analysis and tandem mass spectrometry

“…MALDI-MS/MS analysis was performed as described by Marley et al [18]. MALDI-MS/MS data were processed with GPS explorer 2.0 for creating MASCOT-searchable files as described [15].…”