Dynamic Calibration Enables High-Accuracy Charge Measurements on Individual Ions for Charge Detection Mass Spectrometry

Abstract

Charge detection mass spectrometry (CDMS) depends on the measurement of the charge induced on a cylinder by individual ions by means of a chargesensitive amplifier. For high-accuracy charge measurements, the detection cylinder is embedded in an electrostatic linear ion trap (ELIT), and the ions oscillate back and forth through the cylinder so that multiple measurements are made. To assign the charge state with a low error rate, the charge of each ion must be determined with an uncertainty (root-mean-square deviation) of around 0.2 elementary charges. We show here that high-accuracy charge measurements can be achieved for large ions by dynamic calibration of the charge measurement using an internal standard. The internal standard is generated by irradiating the detection cylinder, by means of a small antenna, with a radiofrequency signal. Using this approach, we have obtained a relative charge uncertainty of around 5 × 10 −4 , allowing charge-state resolution to be achieved for single ions with up to 500 charges. In another application of this approach, the detection cylinder is irradiated with a signal that counteracts the transients generated when the potentials on the ELIT end-caps are switched to trapping mode. Using this approach, the dead time after switching (during which the signal cannot be analyzed) has been reduced by more than an order of magnitude. With chargestate resolution for ions with up to 500 charges, we were able to calibrate the charges precisely. The results show that the response of the charge-sensitive amplifier with dynamic calibration is linear to within a small fraction of an elementary charge.

Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry

“…Here, we report the realization of that potential with isotopic resolution for tetrameric pyruvate kinase (232 kDa) and tetrameric β-galactosidase (466 kDa), both of which were previously analyzed using ion trap CDMS. 18,19 This work contributes to the decades-long evolution of mass spectrometry 20 and will help future researchers obtain greater clarity on desolvation and molecular composition for ions and biological complexes of ever higher mass.…”

“…Since CDMS has been used to characterize megadalton-sized analytes, , the potential of I 2 MS to set new resolution limits for native mass spectrometry is clear. While CDMS has already been utilized to improve the resolving power of ion traps, , the massive disparity between ion traps and FT instruments in terms of resolving power suggests that the full potential of individual ion workflows is largely unrealized. Here, we report the realization of that potential with isotopic resolution for tetrameric pyruvate kinase (232 kDa) and tetrameric β-galactosidase (466 kDa), both of which were previously analyzed using ion trap CDMS. , This work contributes to the decades-long evolution of mass spectrometry and will help future researchers obtain greater clarity on desolvation and molecular composition for ions and biological complexes of ever higher mass.…”

Higher Resolution Charge Detection Mass Spectrometry

“…We recently reported studies where this goal had been achieved for ions with up to 500 charges. 53 When charge states can be assigned with a low error rate, the mass resolving power is determined almost entirely by the uncertainty in the m/z.…”

“…65 Efforts to improve the m/z resolving power of CDMS ELITs have focused on reducing the dependence of the ion's oscillation frequency on the ion energy and the ion's radial offset and angular divergence. 66 In this manuscript we describe studies where recent improvements in the accuracy of the charge measurements in CDMS 53 are combined with higher resolution m/z measurements 66 to obtain a mass resolving power almost an order of magnitude better than that achieved in previous CDMS measurements. The improved resolution is demonstrated by monitoring the assembly reaction of hepatitis B virus (HBV) capsids under different initial assembly conditions.…”

UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data

“…[9][10] Custom instruments have achieved the highest charge resolution with uncertainties less than a single charge, but charge resolution can still limit the measurements in some cases. 1,4,16 Here, we address this challenge in charge uncertainty with UniDecCD (UCD), which provides the first deconvolution algorithm for CD-MS data. UCD builds on our widely used UniDec software for deconvolution of ESI-MS data, and translates the UniDec algorithm to CD-MS data.…”