A new ion mobility-linear ion trap instrument for complex mixture analysis

Abstract

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.

Figure 1

Schematic representation of the IMS-MS instrument showing the funnels (F), gates (G), and ion activation regions (IA). The boxed portion of the schematic shows the LTQ Velos (ThermoScientific) mass spectrometer consisting of rf focusing devices, the linear ion trap mass analyzer, and the ETD source.

Figure 2

(A) Two-dimensional (2D) t_D (m/z) heat-plot of a cytochrome c tryptic digest containing the phosphorylated peptide standard [KRPsQRHGSKY-NH₂ (m/z 475.2)]. The color map for the plot is shown on a logarithmic scale ranging from −2 to 3 to show the low-intensity [M + H]⁺ ions. (B) Expanded region of a 2D t_D (m/z) heat-plot for the same digest sample upon performing IMS-CID. The same color scale is used. The XIDTDs of the precursor and neutral loss product ions are shown as insets on the respective 2D plots. An asterisk denotes the location of the precursor ion both 2D plots. (C and D) Mobility-selected ETD spectra of the precursor and neutral loss ions, respectively. Assigned fragment ions are labeled. The $z_8^{\ast }$ label denotes the fragment ion corresponding with the S4 residue for neutral loss of H₃PO₄ by IMS-CID.

Figure 3

(A) 2D t_D (m/z) heat-plot of one replicate analysis of the EXP24 metabolite sample. The color map for the plot is shown on a logarithmic scale ranging from −2 to 3 in order to show low-intensity features. (B) Multistage tandem mass spectrometry (MSⁿ) of the precursor ion of m/z 518.3. The MS³ spectrum (top) is obtained upon activation of the major product ion (m/z 459.2) generated during MS/MS (data not shown). The bottom spectrum shows MS⁴ fragments produced by selecting the ion of m/z 313.2 from MS³ experiments. The peak marked with an asterisk indicates the m/z selected for subsequent MS analysis.

Figure 4

Single replicate XIDTDs of the ion of m/z 518.3 for the CNTRL24 (solid trace) and the EXP24 (dashed-line trace) samples. Each distribution is normalized such that the total area under the curve is unity. The arrow indicates an intensity difference in spectral features between the two samples that was recognized originally in the pixel-by-pixel intensity comparison of the 2D IMS-MS data sets (see text for details).

Figure 5

(A) 2D) t_D (m/z) heat-plot of the EXP24 sample obtained from an IMS-CID-MS experiment. The color map for the plot is shown on the same scale. (B) XIDTDs for the precursor (top) and fragment (bottom) ions of m/z 534.2 and 475.0, respectively. Dashed lines in panel A show the XIDTD regions. (C) Percent difference plots obtained upon comparison of the XIDTD for the fragment ion of m/z 475.0 and the XIDTDs of precursor ions of m/z 534.2 (solid trace) and 496.3 (dashed-line trace).