We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

Using an integrated genomic and proteomic approach, we have investigated the effects of carbon source perturbation on steady-state gene expression in the yeast Saccharomyces cerevisiae growing on either galactose or ethanol. For many genes, significant differences between the abundance ratio of the messenger RNA transcript and the corresponding protein product were observed. Insights into the perturbative effects on genes involved in respiration, energy generation, and protein synthesis were obtained that would not have been apparent from measurements made at either the messenger RNA or protein level alone, illustrating the power of integrating different types of data obtained from the same sample for the comprehensive characterization of biological systems and processes. Molecular & Cellular Proteomics 1:323-333, 2002.The concept of discovery science, best illustrated by the human genome project (1, 2), involves the identification of the components of a system without the prior formulation of hypotheses as to how these components function (3). This scientific method has spawned what has become known as the "systems" approach to biology, which involves the comprehensive characterization of the components of a biological system (i.e. DNA, RNA, and proteins) as a whole, leading to insights into the responses of these components because of systematic perturbations to the system. The objective of the systems biology approach is to identify markers and mechanisms that are important to the function of the perturbed system, with the ultimate goal of developing computational models that enable the prediction of the response of the system to any given perturbation.Traditionally, studies measuring the effects of systematic perturbations have been carried out at the level of transcribed mRNA, most commonly using cDNA arrays and chip technologies (4 -7), or alternative methods for mRNA analysis such as serial analysis of gene expression (8), differential display (9), and cDNA fingerprinting (10). These technologies have been used to distinguish diagnostically between cell types (7,(11)(12)(13)(14)(15) and to differentiate between states (metabolic, activation, pathological) of a particular cell type (6, 16), as well as for the comprehensive analysis of cellular pathways and processes by targeted perturbations of cells (16 -19).Although the measurement of transcribed mRNA has proven to be very powerful in the discovery of molecular markers and the elucidation of functional mechanisms, alone it is not sufficient for the characterization of biological systems as a whole. This is based on several observations. First, comparison of absolute mRNA transcript abundances measured by serial analysis of gene expression (8) with the corresponding protein abundances expressed in exponentially growing Saccharomyces cerevisiae cells has shown that in many cases mRNA abundance is not a reliable indicator of corresponding protein abundance (20), and studies in other systems have reached similar conclusions (21, 22). Furthermore, attenuation...

The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.

We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S. P.; et al. Nat. Biotechnol. 1999, 17, 994-9.). Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.