Comparative proteomic approaches using isotopic labeling and MS have become increasingly popular. Conventionally quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was

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... tabolically labeled with [ 13 C 6 ]lysine and [ 15 N 4 ]arginine; the other set was unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity, and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a 10-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated, and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG108 cells. Molecular & Cellular Proteomics 5:401-411, 2006. In recent years, various strategies based on stable isotope coding and MS have been established and have become increasingly popular as alternatives to two-dimensional PAGE-based methods (1, 2) for quantitative proteomics (3-8). In these methods, proteins or peptides from different samples are differentially labeled using stable isotopes, and protein quantification is achieved by comparing their relative abundances in MS. Stable isotopes can be introduced through chemical reactions or metabolic incorporation during cell culture. Although both strategies have characteristic strengths and limitations (6), metabolic labeling seems to have gained more attention recently (4, 5, 8, 9) . The labeling process is straightforward and highly efficient. Unlabeled and labeled samples can be combined directly after, or sometimes even prior to, cell lysis and treated as a single sample in all subsequent steps, thus minimizing errors introduced during sample preparation. In most comparative proteomic studies, differentially labeled peptides are analyzed by LC-MS/MS in a data-dependent manner in which MS/MS scans are automatically triggered after MS survey scans. In these experiments, MS/MS data are used for protein identification, and MS data are used for quantification. Protein ratios can then be determined by comparing the relative intensities of MS signals from differentially labeled peptides. Alternatively quantification can be based on extracted ion chromatograms (XICs) 1 of the MS signal. However, both MS and XIC signals are often strongly affected by background noise, which can limit quantification accuracy and dynamic range (10, 11) especially for certain types of mass spectrometers such as ion traps (11). Background noise is especially problematic in quantitative experiments because the relative contribution of even a constant background signal is greater for low abundance peptides than for higher abundance peptides. In addition, when samples are of high complexity, co-eluting isobaric peptides or contaminants can interfere with target signals. An alternative strategy is to use MS/MS data for quantification. Although much less frequently used than MS-based strategies, MS/MS-based quantification offers great potential for enhancing accuracy and dynamic range because MS/MS data generally have much better s/n than XIC and MS data (9, (11) (12) (13) . Especially in ion traps, small signals with low s/n can be accumulated in the trap before fragmentation to achieve MS/MS spectra with much higher s/n than MS spectra (11). Several previous studies have used this strategy to measure protein ratios. In two recent reports, peptides were labeled with isobaric tags that contain labile bonds easily cleaved