The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined. Protease catalyzed 18 O exchange is a simple method to differentially label peptides, but the lack of robust software tools to analyze the data from mass spectra of 18 O labeled peptides generated by common ion trap mass spectrometers has been a limitation. ZoomQuant is a stand-alone computational tool that analyzes the mass spectra of 18 O labeled peptides from ion trap instruments and determines relative abundance ratios between two samples. Starting with a filtered list of candidate peptides that have been successfully identified by Sequest, ZoomQuant analyzes the isotopic forms of the peptides using high-resolution zoom scan spectrum data. The theoretical isotope distribution is determined from the peptide sequence and is used to deconvolute the peak areas associated with the unlabeled, partially labeled, and fully labeled species. The ratio between the labeled and unlabeled peptides is then calculated using several different methods. ZoomQuant's graphical user interface allows the user to view and adjust the parameters for peak calling and quantitation and select which peptides should contribute to the overall abundance ratio calculation. Finally, ZoomQuant generates a summary report of the relative abundance of the peptides identified in the two samples. One of the main goals of many proteomics experiments is to determine the difference in abundance of proteins between samples representing different biological conditions. Analyses of the peptides from the two samples as independent LC/MS runs can have difficulties related to issues of nonlinearity of chromatography, reproducibility of peptide coverage due to timing of data dependent sampling by the MS, and ion suppression in complex samples. To overcome these limitations, a number differential stable isotope labeling methods have been developed so that labeled peptides from one sample can be combined with unlabeled or differently labeled peptides from a second sample and both samples analyzed in a single analytical run [1][2][3][4][5][6][7][8][9]. This approach overcomes the problems of timing and reproducibility between the analytical runs and provides an accurate determination of the ratio of abundance of proteins between the two samples.Differential peptide labeling for analysis by mass spectroscopy relies on introducing a mass difference using stable isotopes and it is assumed that this isotopic difference does not alter the peptides' chromatographic or fragmentation properties. Labeling can take place biosynthetically, using media containing 15 N amino acids for example [1], after sample isolation by introducing a chemical modification using the ICAT reagent [2-4], or by exchanging atoms for a heavier isoto...
Stable isotope labeling with 18 O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of 18 O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers. In this paper, we describe a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using 18 O. The method utilizes zoom scans to provide high resolution data. This allows for accurate calculation of 18 O/ 16 O ratios for peptides even when as much as 50% of a 18 O labeled peptide is present as the singly labeled species. The use of zoom scan data also provides sufficient resolution for calculating accurate ratios for peptides of +3 and lower charge states. Sequence coverage is comparable to that obtained with data acquisition modes that use only MS and MS/MS scans. We have employed a newly developed analysis software tool, ZoomQuant, which allows for the automated analysis of large data sets. We show that the combination of zoom scan data acquisition and analysis using ZoomQuant provides calculation of isotopic ratios accurate to ~21%. This compares well with data produced from 18 O labeling experiments using time of flight (TOF) and Fourier transform-ion cyclotron resonance (FT-ICR) MS instruments.There is a growing impetus to develop methods for relative quantification of proteins and peptides that are compatible with shotgun methods and high throughput experiments. Two general approaches have thus far been used. Both approaches depend on the use of "light" and "heavy" isotopes to differentially label proteins from two different populations of cells grown under variant conditions. The resultant mass shift provides a mass based separation for otherwise identical molecules from both cell populations that can be used for relative quantification. In-vivo labeling methods, such as metabolic labeling relying on the incorporation of isotopically labeled specific amino acids [1], or complete substitution of 14 N with 15 N [2], cannot be used for many mammalian systems. Yates et al. have reported success with in-vivo metabolic labeling of rat proteins by providing them with a diet enriched in 15 N labeled amino acids [3]. Post-translational methods of labeling are applicable to eukaryotic systems. These methods introduce mass shifts through the modification of the side chains of specific amino acids, such lysine [4] and cysteine [5], labeling of the carboxy, or amino terminus of peptides. Isotope-coded affinity tag (ICAT), which introduces "heavy" and "light" variants of an affinity tag that modifies cysteines, is the most well developed technology for post translational modification of peptides for differential protein expression analysis b y mass Experimental ChemicalsEquine s...
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