The goal of quantitative proteomics is to examine the expression levels of all of the proteins in a biological system and recognize those that change as a function of some stimulus. Quantification is now frequently based on derivatization of peptides with isotopically distinguishable labeling agents. This study examines the extent to which isotopic forms of peptides having the same amino acid sequence are resolved by reversed-phase chromatography and assesses the degree to which resolution of these isotopically different forms of a peptide impact quantification. Three derivatizing agents were examined, the do and d3 forms of N-acetoxysuccinimide, the do and d4 forms of succinic anhydride, and the do and d8 forms of the commercial ICAT reagent Peptide mixtures from control and experimental samples were derivatized individually, mixed, subjected to reversed-phase chromatography, and analyzed by ESI-MS. When partial resolution of the isotopic forms of a peptide occurs, the largest error in assessing the true isotope ratio in a sample occurs when sampling at the extremes of a peak. Early in the elution of a peak, the sample will be enriched in the deuterated species, whereas the opposite is true at the tailing edge of a peak. Acetylated peptides showed the lowest degree of separation. Resolution of the deuterated and nondeuterated forms in this case was 0.023. This amounts to slightly over a 1-s difference in their peak maxima and can cause a typical error of +/- 6% at the leading and tailing edges of a peak. In contrast, resolution of the deuterated and nondeuterated forms of the ICAT reagent were calculated to be 0.45. This means that in a peak of 1-min width (W1/2), the peak maxima will vary by approximately 30 s, and measurement errors of -83 and +500% can occur at the leading and tailing edges of a peak. It is concluded that resolution of isotopic forms of a peptide can cause substantial quantification errors in quantitative proteomics.

A miniaturized two-electrode electrochemical (EC) cell was developed and was coupled on-line with an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-FTICR MS). Electrochemistry on-line with mass spectrometry, EC/ESI-FTICR MS, of triphenylamine (TPA), which undergoes one-electron oxidation to form a radical cation (TPA*+), demonstrates a significant sensitivity enhancement compared to ESI-FTICR MS. The on-line EC cell configuration with a stainless steel ES needle as the working electrode produces the highest sensitivity in EC/ESI-MS. The results provide evidence that, during the ES ionization, electrolytic reactions occur mainly in the ES tip region, as previously predicted. The results demonstrate that ESI-MS signal suppression by tetrabutylammonium perchlorate electrolyte, which can be a problem, is minimized in EC/ESI-MS. TPA*+ dimer tetraphenylbenzidine (TPB) can be detected by EC/ESI-MS, together with TPA*+, as TPB*+ and TPB2+. The high mass resolving power of FTICR MS was exploited to identify TPB2+ dication in the presence of [TPA*+ - H*]+ ions of the same m/z, from their respective isotopic distributions. The dimer dication TPB2+ can be detected only in EC/ESI-MS.

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.

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