Acid-Labile Isotope-Coded Extractants:  A Class of Reagents for Quantitative Mass Spectrometric Analysis of Complex Protein Mixtures

Abstract

Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.

Quantitative mass spectrometry in proteomics: a critical review

“…Following the initial success of the ICAT approach, several variations on this chemical reagent class emerged to improve, e.g., recovery of labeled peptides or chromatographic properties [28][29][30][31]. Other thiol-specific reagents typically contain halogen-substituted carboxylic acids or amides [32][33][34][35] or employ the Michael-type addition reaction to carbonyl groups (e.g., maleiimide esters and vinylpyridine) [36,37]. As cysteine is a rare amino acid, ICAT and related methods significantly reduce the complexity of the peptide mixture which can be advantageous when highly complex samples are analyzed.…”

Find Pairs: The Module for Protein Quantification of the PeakQuant Software Suite

“…ICAT: Isotope-Coded Affinity Tags: e.g., 8 Da (Gygi et al, 1999) ALICE: Acid-Labile Isotope-Coded Extractants: 1 Da (Qiu et al, 2002) 2-Methoxy-4,5-dihydro-1 H-imidazole: 4 Da (Peters et al, 2001) MCAT: Mass-Coded Abundance Tagging: 42 Da (Cagney and Emili, 2002) GIST: Global Internal Standard Technology: 4 Da (Chakraborty and Regnier, 2002) 18 O/ 16 O: 4 Da (Schnolzer et al, 1996;Yao et al, 2001) ICPL: Isotope Coded Protein Label: 6 Da (Schmidt et al, 2005) iTRAQ: isobaric Tag for Relative and Absolute protein Quantitation, reporter ions in MS/MS spectra: e.g., 114, 115, 116, and 117 Da (Ross et al, 2004) TMT: Tandem Mass Tags, reporter ions in MS/MS spectra (Thompson et al, 2003) SILAC: Stable Isotope Labeling with Amino Acids in Cell Culture, e.g., with deuterated leucine (Leu-d3) (Foster et al, 2003;Ong et al, 2002); or with heavy lysine and/or arginine (Andersen et al, 2005;Ibarrola et al, 2003); or with heavy methionine (Ong et al, 2004) 14 N/ 15 N: variable additive mass shift; it can be estimated by a database-averaged multiplicative mass shift or mass shift factor (MSF): 1.012198 (Oda et al, 1999;Skirycz et al, 2011). MSF values can be used to detect peak pairs without any knowledge about the protein sequence.…”

Mass spectrometry in aging research

“…Therefore, recent improvements of ICAT reagents include the design of a solidphase isotope tagging method, avoiding biotin, and incorporation of a photocleavable linker (structure 3); after solid-phase purification of ICAT-labeled peptides and photolysis, the remaining peptides essentially contain an additional normal or heavy isotope-containing Leu residue (Zhou et al, 2002b). Instead, Qiu et al (2002) designed an acid-labile isotope-coded extractant (ALICE; structure 4).…”