Valet Parking for Protein Ion Charge State Concentration: Ion/Molecule Reactions in Linear Ion Traps

Abstract

There are several analytical applications in which it is desirable to concentrate analyte ions generated over a range of charge states into a single charge state. This has been demonstrated in the gas phase via ion/ion reactions in conjunction with a technique termed ion parking, which can be implemented in electrodynamic ion traps. Ion parking depends upon the selective inhibition of the reaction of a selected charge state or charge states. In this work, we demonstrate a similar charge state concentration effect using ion/molecule reactions rather than ion/ion reactions. The rates of ion/molecule reactions cannot be affected in the manner used in conventional ion parking. Rather, to inhibit the progression of ion/molecule proton transfer reactions, the product ions must be removed from the reaction cell as they are formed and transferred to an ion trap where no reactions occur. This is accomplished here with mass-selective axial ejection (MSAE) from one linear ion trap to another. The application of MSAE to inhibit ion/molecule reactions is referred to as "valet parking" as it entails the transport of the ions of interest to a remote location for storage. Valet parking is demonstrated using model proteins to concentrate ion signal dispersed over multiple charge states into largely one charge state. Additionally, it has been applied to a simple two-protein mixture of cytochrome c and myoglobin.

Figure 1.

Ion optics and essential elements of the QTRAP 4000 ion path.

Figure 2.

Schematic of the external manifold used to introduce reagent in q2 for ion/molecule reactions.

Figure 3.

Positive electrospray mass spectrum of ubiquitin with a) no additional storage time in q2 or Q3, b) 400 ms storage in q2 (i.e. 400 ms ion/molecule reaction), and c) 400 ms storage in Q3 prior to mass analysis. The abundance weighted average charge state is represented with the colored dashed line.

Figure 4.

Positive electrospray mass spectrum of ubiquitin a) pre-ion/molecule reaction, b) post ion/molecule reaction, and c) valet parking of the 8+ charge state. The ion/molecule reaction time is 900 ms, the IQ3 barrier was set to 1.5 V, and a valet parking waveform of 1.9 V at 114.367 kHz was used.

Figure 5.

Positive electrospray mass spectrum of cytochrome c a) pre-ion/molecule reaction, b) post ion/molecule reaction, and valet parking of the c) 12+, d) 11+, or e) 10+ charge state. The ion/molecule reaction time is 900 ms, the IQ3 barrier was set to 1.8 V, and a valet parking waveform of 1.8 V at 114.367 kHz was used.

Figure 6.

Positive electrospray mass spectrum of myoglobin a) pre-ion/molecule reaction, b) post ion/molecule reaction, and valet parking of the c) 16+, d) 15+, or e) 14+ charge state. The ion/molecule reaction time is 600 ms, the IQ3 barrier was set to 2.0 V, and a valet parking waveform of 2.0 V at 114.367 kHz was used.

Figure 7.

Positive electrospray mass spectrum of a cytochrome c and myoglobin mixture a) pre-ion/molecule reaction, b) post ion/molecule reaction, and c) valet parking of the myoglobin 13+ charge state. The ion/molecule reaction time is 900 ms, the IQ3 barrier was set to 2.0 V, and a valet parking waveform of 2.0 V at 114.367 kHz was used.

Figure 8.

Calculated ion frequencies and ion frequency dispersion for consecutive charge states of cytochrome c when cytochrome c 12+ is placed at a) q = 0.4 and b) q = 0.8.