FT-ICR MS optimization for the analysis of intact proteins - PubMed (original) (raw)

FT-ICR MS optimization for the analysis of intact proteins

Aleksey V Tolmachev et al. Int J Mass Spectrom. 2009.

Abstract

Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) remains the technique of choice for the analysis of intact proteins from complex biological systems, i.e. top-down proteomics. Recently, we have implemented a compensated open cylindrical ion trapping cell into a 12 T FT-ICR mass spectrometer. This new cell has previously demonstrated improved sensitivity, dynamic range, and mass measurement accuracy for the analysis of relatively small tryptic peptides. These improvements are due to the modified trapping potential of the cell which closely approximates the ideal harmonic trapping potential. Here, we report the instrument optimization for the analysis of large macro-molecular ions, such as proteins. Single transient mass spectra of multiply charged bovine ubiquitin ions with sub-ppm mass measurement accuracy, improved signal intensity, and increased dynamic range were obtained using this new cell with increased post-excitation cyclotron radii. The increased cyclotron radii correspond to increased ion kinetic energy and collisions between neutrals and ions with sufficient kinetic energy can exceed a threshold of single collision ion fragmentation. A transition then occurs from relatively long signal lifetimes at low excitation radii to potentially shorter lifetimes, defined by the average ion-neutral collision time. The proposed high energy ion loss mechanism is evaluated and compared with experimental results for bovine ubiquitin and serum albumin. We find that the analysis of large macro-molecules can be significantly improved by the further reduction of pressure in the ion trapping cell. This reduces the high energy ion losses and can enable increased sensitivity and mass measurement accuracy to be realized without compromising resolution. Further, these results appear to be generally applicable to FTMS, and it is expected that the high energy ion loss mechanism also applies to Orbitrap mass analyzers.

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Figures

Figure 1

Figure 1

ESI mass spectrum of ubiquitin obtained using the compensated cell, with an excitation attenuation of 7 dB, from a single transient 1.3 s long, with resolution of ~230,000 and insert detail of the 11+ charge state.

Figure 2

Figure 2

Total ion current (TIC) versus excitation power (plotted in terms of attenuation, dB) for the compensated cell configuration, spectra were acquired at ~1 × 10−10 Torr (squares), open cell configuration, ~1 × 10−10 Torr (triangles), and compensated cell configuration at an increased pressure of ~3 × 10−10 Torr (circles).

Figure 3

Figure 3

Mass measurement error, plotted as ppm, versus excitation power, plotted in terms of attenuation dB, for the compensated cell configuration (squares) and open cell configuration (triangles). The internal calibration using 6 charge states of ubiquitine was used for the mass measurement error calculation.

Figure 4

Figure 4

Resolution of ubiquitin 7 to 13+ obtained at excitation attenuations of 9 dB (circles), 7 dB (diamonds), 5 dB (triangles), and 3 dB (squares) corresponding to approximately 0.4, 0.5, 0.6, and 0.75 of the maximum cell radius, Rmax, of ~3.0 cm.

Figure 5

Figure 5

Transient decay curves showing the intensity of the most abundant isotope peak for ubiquitin 7 to 13+ charge states versus time obtained by Fourier-transforming the full 1 M word transient in eight 128 k word segments. Each transformed segment measures the ion signal obtained at a certain time point in the transient. The observed segment intensities were normalized to the intensity of the first segment for each charge state. Plots are shown for mass spectra obtained at excitation attenuations of a) 9 dB, b) 7 dB, c) 5 dB and d) 3 dB for ubiquitin 7+ (open triangles), 8+ (open circles), 9+ (open squares), 10+ (solid circles), 11+ (diamonds), 12+ (solid triangles), and 13+ (solid squares).

Figure 6

Figure 6

The characteristic signal lifetime, τ, for ubiquitin charges states 7 to 13+ (plotted as 1/z) calculated by fitting the transient decay curves (see Figure 5) observed at 7 dB (diamonds), 5 dB (triangles), and 3 dB (squares) to an exponential function. Results from a pressure of ~1 (triangles pointing up) and 3 (triangles pointing down) × 10−10 Torr are shown for an excitation attenuation of 5 dB. Pressure for all others was ~1 × 10−10 Torr.

Figure 7

Figure 7

The characteristic signal lifetime, τ, at different excitation power levels (in terms of attenuation dB) for ubiquitin 8+ (open circles), 9+ (squares), 10+ (solid circles), at a pressure 3 × 10−10 Torr.

Figure 8

Figure 8

LC-MS mass spectra of bovine serum albumin (BSA) obtained by summing 50 scans of the BSA LC peak. Several isoforms with various adducts were observed and the base peak of the 63+ cluster is shown in the insert.

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