We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1⌬ and xrn1⌬ mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5 to 3 decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression. Molecular & Cellular Proteomics 3:1154 -1169, 2004.An initial step in the systematic investigation of cellular processes is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of multiple states (e.g. several experimental controls or time-course studies) difficult to undertake. Multiple 2-plex datasets can be combined after separate analyses, but there is a high likelihood that different sets of peptides and proteins will be identified between each experiment. In addition, the use of massdifference labels increases MS complexity, and this problem increases with numbers of a multiplexed set. Finally, the cysteine-selective affinity strategy for reduction of sample complexity (ICAT) is not amenable to identification of post-translationally modified peptides, as the majority of posttranslational modification (PTM) 1 -containing peptides are discarded at the affinity step.We have developed a multiplexed set of reagents for quantitative protein analysis that place isobaric mass label...

We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.

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Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ® derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ®-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27±5.22, 21.18±10.94, and 18.34±14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39±5.35, 6.23±3.84, and 35.37±29.42. Both GC-MS and iTRAQ®-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.

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