The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and 14N/15N metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the 14N/15N metabolic labeling strategy, we achieve 98% 15N incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically g...

Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry.

The number of publications documenting the utility of electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for the analysis of biological molecules has increased in geometric proportion spanning diverse areas of research. Currently, we are investigating the capabilities of ESI-FTICR to quantify relative molecular ion abundances of biopolymers, an area which has not been explored rigorously. We present here the results of an investigation of a two-component system utilizing equine heart cytochrome c (EH) as the analyte and bovine heart cytochrome c (BH) as a constant concentration internal standard. As these compounds are relatively large ( approximately 12 kDa), they will become multiply charged during the electrospray process. Using appropriate solution and instrument conditions, the 7(+) and 8(+) charge states were enhanced for both cytochrome c species. We report that using the average of the ion abundances for the two charge states observed for each species, the linear curve (intensity ratio vs concentration ratio) had a dynamic range of 0.045-2.348 microM (1.7 orders of magnitude). Linear least-squares regression analysis (LLSRA) of these averaged ion abundances (i.e. [(EH + 7H(+))(7+)/(BH + 7H(+))(7+) + (EH + 8H(+))(8+)/(BH + 8H(+))(8+)]/2) yielded the equation y = 1.005x + 0.027. The slope of the line with its calculated precision, reported as one standard deviation, is 1.005 +/- 0.0150, which is statistically ideal (i.e. equal to unity). However, LLSRA of the ion abundances of the two individual charge states were significantly different (i.e. the slope of the (EH + 7H(+))(7+)/(BH + 7H(+))(7+) peak intensity ratio vs molar ratio data was 0.885 +/- 0.0183 and the slope of the (EH + 8H(+))(8+)/(BH + 8H(+))(8+) data was 1.125 +/- 0.0308). We attribute this difference to the variation in primary amino acid sequence for the two cytochrome c species. Both have 104 amino acids, but there are three residue substitutions between EH and BH; one of the substitutions confers an additional basic site to EH. While this extra basic residue may imply an additional charging site, the low charge states observed under the solution conditions employed indicate that most (>66%) basic sites are not protonated. However, the extra basic site also renders EH slightly more hydrophilic. These results present significant considerations when choosing internal standards for the quantification of large proteins by ESI-FTICR-MS and demonstrate that relative molecular ion signals in FTICR can be used to quantify macromolecular species in the nanomolar regime.

Hydropathic influences on the quantification of equine heart cytochrome c using relative ion abundance measurements by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

Thermochemical properties of halogen-substituted methanes, methyl radicals, and carbenes in the gas phase

Fundamental research into the quantitative properties of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has yielded interesting observations, especially in terms of factors affecting the accuracy of relative ion abundances. However, most of the previous discussions have focused on theoretical systems, or systems of limited scope. In this paper, we document ion motion attributes of a 30 spectra (six samples, five replicates each) system previously established as linear over two orders of magnitude. Observed behaviors include the perturbation of one charged species (cyclosporin A, CsA) of low ion density to a cyclotron orbit of greater radius than that of an almost identical, but slightly mass-separated species (CsG) with a higher ion density. This radial perturbation is attributed to the coulombic repulsion between the two ion clouds as they interact during the excitation process, as previously proposed by Uechi and Dunbar. Magnitudes of the perturbation were confirmed by making cyclotron radii determinations utilizing the ratio of the third-to-first harmonics for the charged species of interest. It was found that these radial differences can account for as much as a 55% signal bias in favor of CsA for a single sample and a >20% positive bias in the slope of the regressed data set. A second behavior noted that also contributes to the potential inaccuracy of relative ion abundance measurements is the difference in signal decay rates for CsA and CsG. Damping constants and initial time domain signal amplitudes were evaluated using segmented Fourier transforms. Discrepancies in decay rates were not expected from two species that have essentially identical collisional cross-sections. However, it has been observed that the faster decay rates are observed by the species of lower ion cloud density. We have attributed this differential signal decay phenomenon to the rates of loss of phase coherence for the two ion clouds. Previously, others have reported that less dense ion clouds are more susceptible to shearing and other disruptive forces during the course of their excited cyclotron motion. Our experimental evidence supports that it is the loss of cloud coherence that accounts for the signal loss over time, with the less dense cloud de-phasing more quickly. As the ion populations of the two investigated species near equivalence, so do their time constants.

Impact of ion cloud densities on the measurement of relative ion abundances in Fourier transform ion cyclotron resonance mass spectrometry: experimental observations of coulombically induced cyclotron radius perturbations and ion cloud dephasing rates

Tremendous developments in mass spectrometry have taken place in the last 40 years. This holds for both the science and the instrumental revolutions in this field. In chemistry the research was heavily focused on organic molecules that upon electron ionization fragmented via complex mechanistic pathways as shown by isotopic labeling experiments. These studies, including ion structure determinations, were performed with use of double focusing mass spectrometers of both conventional and reversed geometry, and equipped with various types of metastable ion scanning and collision-induced dissociation techniques developed by physical and analytical chemists. Time-resolved mass spectrometry by use of the field ionization kinetics method, developed by physical chemists, was another powerful way to unravel details of unimolecular gas phase ion dissociations. Then the development of new ionization methods, such as desorption chemical ionization, field desorption, and fast atom bombardment permitted not only to analyze unvolatile, thermally labile and higher molecular weight compounds, but also to study their chemical behavior in the gas phase, initially with use of double focusing instruments and later on with multisector and hybrid mass spectrometers. These ionization methods also enabled to study organometallic compounds and increasingly the field of medium-sized to large biomolecules, the latter being exploded in the last decade by the development of electrospray- and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Another area of research concerned the bimolecular chemistry of organic ions with organic molecules in the gas phase. Initially this was performed with use of among others drift-cell ion cyclotron resonance spectroscopy, that later on was replaced by the developed method of ion trapping and Fourier transform ion cyclotron resonance. Combination of the latter with the afore-mentioned ionization methods has shifted also in this case the research on organic molecules to organometallic/inorganic systems, and predominantly to biomolecules in the last decade. This invited review will describe the research efforts made by the author's group over the last 40 years together with some personal experiences during his career.

Four decades of joy in mass spectrometry

Segmented Fourier transform and its application to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry: ion abundances and mass measurements

Christ W.F. Kort

Papers

Segmented Fourier transform and its application to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry: ion abundances and mass measurements