After optimizing the instrument conditions, we evaluated the utility of this hybrid instrument with the analysis of peptide mixtures. The initial attempt was to separate three standard peptide ions, i.e., doubly charged bradykinin (m/z 530), doubly charged angiotensin II (m/z 523), and triply charged neurotensin (m/z 558), with dual-gate filtration experiments. The selected mobility scanning mode was utilized to predict the scan window, which was determined to be 46–51 ms. Then the mobility separation experiments were set up using the mobility scanning dual-gate mode with the following parameters: 2 s h2 accumulation time, 39 dual-gate pulses per h2 accumulation cycle, 100 data averages, 0.2 ms step increment, 12.5 kV nanospray voltage, 10 kV applied to the first ring electrode, 150°C drift tube heating temperature (measured drift space temperature was 125°C), and air used as drift gas. Figure 5 shows the reconstructed mobility spectra of these three ions with gate pulse width at 0.5 ms (Fig. 5(a)) and 0.4 ms (Fig. 5(b)). It should be noted the same pulse width was applied to both gates. As shown in Fig. 5, bradykinin was baseline-resolved from neurotensin and angiotensin II; neurotensin and angiotensin II were partially resolved. A pulse width of 0.4 ms showed slightly better resolution, but further decrease in gate pulse width resulted in significantly reduced ion signals. Given the nature of dual-gate mobility experiments, the effective drift length (L) and drift voltage (V) are the distance and potential difference between the first gate and the second gate, respectively, since once ions exit the second gate, the drift time measurement is completed. We calculated the reduced mobility constants (K0) for these three ions using the equation:
with L ∼26.5 cm, V 8.3 kV, T (temperature) 398 K, and P (pressure) 700 Torr. The measured K0 values for all three ions are listed in Table 1. The measured K0 values for doubly charged bradykinin and angiotensin II ions in this study were 1.12 and 1.08, respectively, which were within 98% of reported literature values.15
One primary advantage of IMS is its capability to separate isomeric compounds, which is unattainable for FTICR-MS in spite of its superior resolving power. Tandem mass spectrometry (MS/MS) fragmentation experiments are often necessary for distinguishing these isomeric compounds. However, LC/MS/MS experiments will not be able to resolve the differences and make correct assignments of fragment ions if two isomers coelute, which is unfortunately true in many cases because of high structural similarities of isomers. Thus, we took further efforts to look into the isomer separation capability of this hybrid instrument. The isomers we chose for this experiment were two peptides with the same sequence but different phosphorylation sites (see Table 1). Protein and peptide phosphorylation is a type of posttranslational modification with great significance in many biological systems, since site-specific phosphorylation can have dramatic effects on system functionality.37 The mobility scanning dual-gate mode was applied to acquire the mobility spectra for each peptide individually and mixtures of these two peptides. The applied parameters for this set of experiments included 2 s h2 accumulation time, 47 dual-gate pulses per h2 cycle, 100 data averages, 37–42 ms scan window, 0.5 ms gate pulse width, 13 kV nanospray voltage, 11.8 kV first ring electrode voltage, drift temperature 125°C, and air as drift gas. The effective drift voltage was 9.7 kV. The resulting mobility spectra for each individual peptide and mixtures of two peptides are presented in Fig. 6, which shows partial separation of the two isomeric phosphopeptides. The calculated K0 values for these two peptides are provided in Table 1. Due to the limited instrument sensitivity, further decrease in gate pulse width resulted in no mobility signal. Therefore, additional improvement of ion transfer efficiency and duty cycle will be essential for achieving baseline separation of these two isomeric phosphopeptides with the use of 0.2 ms or lower gate pulse width.