Comparative proteomics based on stable isotope labeling and affinity selection.

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Potential of silica monolithic columns in peptide separations.

The objective of the work described here was to evaluate the efficacy of silica monolith supports in high-speed reversed-phase liquid chromatography (RPLC) of peptides. This was done using a commercial Chromolith column with an octadecylsilane stationary phase and a tryptic digest of cytochrome c. Columns (100 mm x 4.6 mm) were operated at mobile phase velocities ranging from 1 ml/min (2.0 mm/s) to 10 ml/min (25 mm/s). There was little noticeable change over this flow rate range in either resolution, peak elution volume, or analyte concentration in collected fractions. It was concluded that capillary columns in this silica monolith format would be particularly valuable in peptide separations for proteomics. There was, however, a small, but perceptible contamination of peaks at high mobile phase velocity with earlier eluting analytes. Based on the fact that peak shape did not change at high mobile phase velocity, it is suggested that this phenomena might be due to the presence of peptide conformers in structural equilibrium on the sorbent surface. When elution rate exceeds the rate of conformer interchange, conformers could elute as broadened or even separate peaks.

Minimizing resolution of isotopically coded peptides in comparative proteomics.

Stable isotopes are now widely used to quantify concentration changes in proteomics. This paper focuses on the resolution of isotopically coded peptides and how isotope effects occurring during chromatographic separations can be minimized. Heavy isotope derivatizing agents used in this work were the commercially available 2H8-ICAT reagent and 13C4-succinic anhydride. The ICAT reagent derivatizes cysteine-containing peptides, whereas the succinic anhydride reacts with primary amine groups in peptides. It was observed during reversed-phase chromatography of peptides from a BSA tryptic digest differentially labeled with the 2Hr and 2H8-ICAT reagents that resolution of the isoforms exceeded 0.5 with 20% of the peptides in the digest. Three-fourths of the peptides in this group contained two cysteine residues and were doubly labeled. Only 23% of the peptides labeled with a single ICAT residue had a resolution greater than 0.4. The resolution of peptides differentially labeled with 13C- and 12C-succinate never exceeded +/- 0.01, even in the case of peptides from the BSA digest labeled with 2 mol of succinate. Because this value is within the limits of the method used to determine resolution, it was concluded the 13C- and 12C-coded isoforms of labeled peptides did not resolve. The isotope ratio in the case of 13C/12C coding could be determined from a single mass spectrum taken at any point in the elution profile. This enabled isotope ratio analysis to be completed early in the elution of a peptide from chromatography columns.

Controlling deuterium isotope effects in comparative proteomics.

This paper focuses on identifying structural features responsible for resolution of heavy isotope coded peptides during reversed-phase chromatography. This was achieved by using labeled coding agents that varied in structure, number of deuterium atoms, placement of deuterium in the coding agent, and the functional group targeted by the reagent. Six coding agents were examined. Deuterated versions of the coding agents studied included succinic anhydride-2H4, acetic acid 2,5-dioxopyrrolidin-1-yl ester-2H3, propionic acid 2,5-dioxopyrrolidin-1-yl ester-2H5, pentanoic acid 2,5-dioxopyrrolidin-1-yl ester-2H9, [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium chloride-2H9, and the commercial ICAT-2H8 reagent. It was found that these labeling agents vary widely in both their absolute and relative contribution to the chromatographic isotope effect. Relative effects were evaluated by normalizing resolution for the number of deuterium atoms in the derivatized peptide. The single, most dominant effect was the placement of deuterium atoms relative to hydrophilic functional groups in the coding agent. It was concluded that the probability of a deuterium atom interacting with the stationary phase of a reversed-phase chromatography (RPC) column and impacting resolution is greatly diminished by placing it adjacent to a hydrophilic group, as explained by solvophobic theory. But peptide size and coding agent size were also seen to correlate inversely with the magnitude of the isotope effect. This effect was explained as being due to the relative size of the coding agent versus that of the coding agent-peptide conjugate.