Quantification of singly charged biomolecules by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry utilizing an internal standard

Abstract

The quantification of Cyclosporin A (CsA) and related biological compounds by various mass spectrometric techniques is well established. It is precisely this fact that makes the use of CsA particularly appealing as a model system in evaluating the quantitative properties of electrospray ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). Utilizing the internal standard methodology, we have generated a linear calibration curve ranging from 5-250 ng/mL CsA described by the equation y = 0.00503x-0.00958, with a %RSD slope of 0.74% and a correlation coefficient (r 0 ) equal to 0.999 2 (N = 30). The internal standard (200 ng/mL Cyclosporin G) was found to be necessary to establish the reported dynamic range, two orders of magnitude based on an experimentally determined detection limit of 2.4 ng/mL CsA, and also significantly improved the overall precision of the method. The data represented in the standard curve were collected as 1.05 s single acquisitions (512 K data points at an ADC rate of 500 kHz) and unapodized prior to the Fourier transformation of the time-domain data. We report that full Hanning apodization has a statistically significant negative effect (F-test at 95% confidence level) on the regression statistics of the curve (i.e. increasing the %RSD slope to 1.33%). Finally, we report that acceptable quantitative properties of an FTICR-MS experiment can be realized as soon as the detection of a second isotopic beat for the targeted species is obtained. This should allow for the practical and effective coupling of on-line separation techniques for quantitative analysis using ESI-FTICR-MS. Copyright # 1999 John Wiley & Sons, Ltd. Received 4 December 1998; Accepted 4 December 1998Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has been demonstrated to have impressive capabilities for the analytical characterization of biological materials. Recent reports have described such varied investigations as highly accurate molecular mass determinations of peptides and proteins, 1-4 high resolution tandem mass spectra of exceedingly large biomolecules (b10 kDa), 2,5,6 and structural verification of 50-to 100-mer DNA and RNA sequences. 7,8 The maturation of this increasingly useful technique in the qualitative analysis of biological compounds is well documented. Interestingly, one area of research that has been comparatively slow to develop is the exploration of electrospray ionization (ESI) FTICR-MS as a quantitative tool. Limbach et al. reported the first experimental demonstration of the potentially achievable detection limit ($177 ions at S/N = 3) and dynamic range (b4 orders of magnitude) for an FTICR mass spectrometer with a 1.875-in cubic trap housed within a 3.0 T superconducting magnet. 9 In a more empirical approach, Padley and co-workers quantified both singly and multiply charged ions according to their absolute peak intensities 10 utilizing ESI and observed linearity of $2-3 orders of magnitude for singly charged lysine (147 Da) and 1.5 or...

Understanding the influence of post‐excite radius and axial confinement on quantitative proteomic measurements using Fourier transform ion cyclotron resonance mass spectrometry

“…Not surprisingly, instrumental parameters that result in a τ ratio nearer to 1 generally have MQF values approaching 1. This result corroborates previous observations that accurate quantification can be achieved if two species have similar signal decay rates 29. Importantly, our results extend this observation to include a wider range of experimental conditions.…”

“…Early investigations revealed that accurate quantification with FT‐ICR‐MS can be realized as long as the second isotopic beat is detected 28, 29. In a study by Gordon and Muddiman of cyclosporin A (CsA) and cyclosporin G (CsG), the authors reported coefficients of variation within ∼10% over the concentration range of 0–250 ng/ml CsA with a constant concentration of 200 ng/mL CsG, with the detection of only ten isotopic beats 29. Moreover, the relative standard deviation of the standard curve (CsA/CsG abundance ratio) was not significantly improved with the detection of more isotopic beats.…”

Comprehensive Analysis of Protein Modifications by Top-Down Mass Spectrometry

“…24, 28, 77 Muddiman and co-workers demonstrated a quantitative relationship between the concentration ratios of two proteins which are 97% identical and their ESI/FTMS peak ratios as well as a linear relationship between the protein concentration and the FTMS peak intensity over ~1.5 orders of magnitude. 78 Kelleher group has developed a method called protein ion relative ratio (PIRR) where the relative ratio of MS signal intensity values were used to calculate the relative amount of modified protein forms 77 and applied it to the global assessment of the combinatorial PTMs of core histones 79 and histone H4. 80 Our group has modified such a PIRR method and quantitatively determined the phosphorylation levels in human, mouse, rat, swine, and primate cTnI 25, 28–30 as well as recombinant mouse cardiac myosin binding protein C (cMyBP-C).…”

Suppression correction and characteristic study in liquid chromatography/Fourier transform mass spectrometry measurements

“…found that the signals of small proteins are suppressed by larger ones. Gordon et al reported that due to ion interaction, the spectral signal intensities do not necessarily reflect true trapped‐ion abundances 1, 5. Additional signal suppression phenomena due to the effects of measuring several peptides are reported in Schmid et al 6.…”

“…Over the past decade or so, liquid chromatography/Fourier transform mass spectrometry (LC/FTMS) has gained increasing popularity for its high mass resolution and mass accuracy which allow for sensitive peptide identification both in LC/MS and tandem mass spectrometry (MS/MS) experiments. As the relationship between actual peptide abundance and the corresponding spectrum intensity is approximately linear,1 FTMS is also widely used in quantitative proteomics and biomarker discovery.…”