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...

In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.

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A conceptually novel approach to protein sequencing involves the generation of ragged-end polypeptide chains followed by mass spectroscopic analysis of the resulting nested set of fragments. We report here on the synthesis and development of a volatile isothiocyanate (trifluoroethylisothiocyanate) that allows the identification of several consecutive residues starting with a few picomoles of peptide. The nested set of peptides is generated simply by adding equal aliquots of starting peptide each cycle and driving both the coupling and cleavage reactions to completion. No additional reagents are required to act as chain terminators and retention of the peptide terminal amine allows for subsequent modification with quaternary ammonium alkyl NHS esters to improve sensitivity. Complex washing procedures are not required each cycle, as reagents and by-products are efficiently removed under vacuum, eliminating extractive loss. Multiple peptide samples can be processed simultaneously, with each degradation cycle completed in 35-40 min. The inherent simplicity of the process should allow for easy automation and permit rapid processing of samples in parallel.

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