Simultaneous quantification and identification using ¹⁸O labeling with an ion trap mass spectrometer and the analysis software application “ZoomQuant”

Abstract

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...

Quantitative characterization of changes in the cardiac mitochondrial proteome during anesthetic preconditioning and ischemia

“…18 O (Cambridge Isotope Laboratories, Andover, MA) according to previously published protocols (12,20). Briefly, precipitated proteins for each treatment were divided equally and resuspended in 250 mM ammonium bicarbonate made with either H 2 16 O or H2 18 O.…”

“…On the basis of these initial findings, we aimed in this study to quantitatively analyze changes in protein levels specific to cardiac mitochondria not only after exposure to the preconditioning agent but after exposure to ischemia and reperfusion as well. Quantitative proteomic approaches using isotopic labeling and mass spectrometry allowed for the relative comparison of protein abundances in cardiac mitochondrial samples from control and isoflurane exposed rats before and after ischemia (12,20).…”

Quantitative Mass Spectrometry-Based Approaches in Cardiovascular Research

“…Olivier and colleagues have developed protocols and analysis tools that allow the efficient quantitative analysis of complex biological samples using this approach. 43,44 Label-Free Quantification Recent developments in the MS instrumentation, extensive advances in bioinformatics, and computing power facilitated alternate approaches to protein quantification by label-free methods. Label-free quantification overcomes the expensive and extensive workflows required in the labeling techniques.…”

Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry

“…Enzyme-catalyzed labeling of peptides provides a comprehensive and global quantitation of the cellular proteome. Enzymatic incorporation of 18 O atoms during proteolytic cleavage, most commonly by trypsin, results in peptides with either one or two 18 O atoms at the carboxy terminus (8,43,74,90,108). The labeled sample is mixed with known amounts of an unlabeled peptide sample (for absolute quantification) or a reference sample (for relative quantification) and analyzed by tandem MS.…”

“…Although there are limitations in the method due to the back exchange of 18 O with naturally occurring 16 O, if sufficient care is taken (109), the method is advantageous over other labeling technologies because of its simplicity and global proteomic quantification both in vivo and in in vitro samples. Our laboratory (41,43) has developed protocols and analysis tools that allow the efficient quantitative analysis of complex biological samples using 18 O labeling. Figure 3 illustrates the common work flow and the analytical software tool ZoomQuant.…”