¹⁸O Labeling: a tool for proteomics

Abstract

An evaluation of the proteolytic labeling and quantification of proteins for diagnostic purposes using trypsin and 18O-enriched H2O is presented. We demonstrate that comparative or relative quantitation can be performed effectively with this approach. We have developed a protocol that allows the conservation of the labeled peptides in natural abundance water without fear of back-exchange providing that pH is sufficiently low to quench the catalytic activity of trypsin, but not so low as to promote chemical back-exchange. Because the labeling efficiency depends on the nature of the peptide, a simple linear relationship between the relative 16O/18O digest buffer mixture content (x) and labeling efficiency (y) does not exist; rather it follows a probability based y = x(2) relationship. As such, the extent of peptide labeling using 16O/18O digest buffer mixture ratios may deviate significantly from that expected based on a linear relationship. The evaluation of the relative Ziptip efficiency indicated a loss in sample recovery as the peptide concentration was reduced using normal conditions, suggesting that there is a limit below which there are diminishing returns. In addition, the adsorptive losses due to Speedvac dry down and recovery indicated modest (20%) losses that may vary widely (0-50%) from peptide to peptide. The in-solution digestion efficiency of standard protein mixtures as a function of concentration revealed a linear decrease with decreasing concentration. This is consistent with enzyme kinetic effects and emphasizes a potential quantitation error that could arise when evaluating differential expression based on peptide detection. The results from our studies demonstrate the power of 18O labeling as an optimization tool for proteomics process development.

pQuant Improves Quantitation by Keeping out Interfering Signals and Evaluating the Accuracy of Calculated Ratios

“…4 Among the most commonly used quantitative strategies are full MS scan-based quantitation methods, such as SILAC (stable isotope labeling with amino acids in cell), 5 15 N-labeling, 6 and 18 O-labeling. 7 In these strategies, proteins are metabolically labeled with stable isotopes, digested into peptides, and then analyzed using liquid chromatography (LC)-MS/MS. Quantitation software tools are designed to extract the intensities of pairs of light (L, unlabeled) and heavy (H, labeled) peptides from full MS scans.…”

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

“…Steps prior to this included the initial splitting of the sample and the subsequent lyophilization. All of these steps are possible sources of small errors [16] and the difference between the actual ratio and the targeted ratio for mixing of the differentially labeled samples. …”

“…In the case of ESI instruments, the magnitude of this small 4 Da mass shift is further reduced by the presence of multiply charged species. Isotopic labeling with 18 O also often produces peptides that are present as a mixture of singly labeled peptides ( 18 O 1 / 16 O 1 ) that result in a 2 Da mass shift, and fully labeled peptides ( 18 O 2 ) that result in a 4 Da mass shift [8,11,12,16]. Accurate calculation of isotopic enrichment, therefore, requires quantification of both the singly labeled species and the doubly labeled species.…”

“…Using this method, peptides that contain only a single 18 O atom can now be included in 18 O/ 16 O calculations. It is also possible to sufficiently resolve isotopic clusters so that +1, +2, and +3 charged tryptic peptides can be included in the analysis and yield accurate isotopic ratios.…”

“…Isotopic ratio calculations were performed with newly developed software, "ZoomQuant" [15] that allows for automated calculation of the isotopic ratios. Prior to the development of this software; isotopic ratio calculation for 18 O/ 16 O isotope labeled data sets that included information from the 18 O 1 16 O 1 mixed species could only be done manually. The development of this method and software now makes it possible to obtain qualitative and quantitative information from differentially labeled protein profiling experiments using ESI-IT instruments.…”