Protein quantification by selective isolation and fragmentation of isotopic pairs using FT-ICR MS

Abstract

Isolation of tryptic peptide ions, along with their differentially labeled analogs derived from an artificial QconCAT protein, is performed using multiple correlated harmonic excitation fields in an FT-ICR cell. Simultaneous fragmentation of the isolated unlabeled and labeled peptide pairs using IRMPD yields specific y-series fragment ions useful for quantification. The mass increment attributed to stable isotope labeling at the C-terminus is maintained in the C-terminal fragment ions, providing multiple measurements of labeled/unlabeled intensity ratios during highly selective detection. The utility of this approach has been demonstrated in the absolute quantification of components of an unfractionated chicken muscle protein mixture. (J Am Soc Mass Spectrom 2008, 19, 973-977 [1]. When modeling cellular pathways and networks it is important to quantify the constituent proteins. Relative quantification methods are designed to compare protein amounts in matched samples [2]. However, these methods are limited to identifying changes in the amount of a protein with respect to a second cellular state and samples vary even within control sets of the same organism [3].When comparing samples analyzed at different times, using different instrumental platforms and within different laboratories, absolute quantification using a universal stable isotope-labeled internal standard of known concentration allows direct comparison. Currently, the most widely used methods rely on the well-established principles of stable isotope dilution techniques, using tryptic peptides as surrogate analytes for the proteins of interest [4J. Chemical synthesis and stable isotope incorporation of individual internal standards allow direct signal comparison, enabling the inference of protein quantities [4J. The production of a collection of internal standards in a single step has been enabled by the design of a DNA construct that is transcribed and translated into a protein concatamer, a technique referred to as QconCAT [5][6][7]. The producAddress reprint requests to Prof. Simon J. Gaskell, University of Manchester, School of Chemistry and Manchester Interdisciplinary Biocentre, John Garside Building, 131 Princess Street Manchester Ml 7DN, UK. E-mail: simon.gaskell@manchester.ac.uk tion of a QconCAT protein thereby enables absolute quantification of multiple proteins using their peptide surrogates encoded within the QconCAT [7]. In a typical QconCAT workflow those proteins for which absolute quantification is required are defined and one or more specific reference peptides (Q-peptides) are chosen for each protein [5]. The DNA coding sequences for these peptides are concatenated, inserted into a vector, and expressed in Escherichia coli to form an artificial QconCAT protein. Growth of the transfected E. coli in stable isotope-containing media yields labeled QconCAT protein [5,6]. To achieve absolute quantification, the QconCAT protein is introduced, in a known amount, into the biological sample of interest and proteins are digested with trypsin to...

Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

“…To circumvent many of the complications posed by large‐scale AQUA‐based quantification studies, we developed the QconCAT approach for multiplexed absolute quantification 22–26. In brief, synthetic genes, optimised for heterologous expression in Escherichia coli , encode a single ORF that is a concatenation of tryptic peptides, each of which acts as an internal standard (a quantotypic or Qpeptide) for a different protein (of course it is equally feasible, or even desirable, to encode two or more peptides to report on each analyte protein).…”

Index-ion Triggered MS2 Ion Quantification: A Novel Proteomics Approach for Reproducible Detection and Quantification of Targeted Proteins in Complex Mixtures

“…Other MS2-based quantitative proteomics approach have been described that use peptide-specific fragment ions for quantification (27, 28, 39). However, in these approaches an enlarged precursor isolation window ( e.g.…”

Strategies and Challenges in Measuring Protein Abundance Using Stable Isotope Labeling and Tandem Mass Spectrometry

“…Internal protein standards can also be obtained by metabolic labeling of organisms, such as E. coli (Hanke et al 2008). Additionally, a single synthesized concatemer protein comprised of peptides from 20 proteins of interest (QconCAT) has been generated to quantify a mixture of proteins (Pratt et al 2006;Johnson et al 2008;Rivers et al 2007;. Taken together, these studies show that the absolute quantitation of peptides and proteins using mass spectrometry is feasible (Brun et al 2009).…”