Advancing Orbitrap Measurements of Collision Cross Sections to Multiple Species for Broad Applications

Abstract

Measurement of collision cross section (CCS), a parameter reflecting an ion’s size and shape, alongside high-resolution mass analysis extends the depth of molecular analysis by providing structural information beyond molecular mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to extract CCSs directly by analysis of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these experiments required isolation of individual charge states of ions prior to CCS analysis, limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by determining CCSs from commonly available mass spectra files as well as estimating CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

“…This method allows simultaneous analysis of multiple different ions or charge states but may result in modest overestimation of CCS owing to space charge effects between charge states, as previously described. 46 Ion injection time was optimized for each charge state by varying the ion injection time and finding the ion injection time that gave the lowest decay rate. As previously described, having too few or too many ions injected at once may cause artificially inflated decay rates which yield over-estimated CCS values.…”

“…As previously described, having too few or too many ions injected at once may cause artificially inflated decay rates which yield over-estimated CCS values. 37,46 The C-trap gas pressure was set low (0.1 to 1.0 values, corresponding to 8E-11 to 1E-10 mBar measuring using the UHV gauge), which was optimized to provide measurable signal decay for each peak. Ion mobility CCSs were collected using a home-built atmospheric pressure drift tube as previously described.…”

“…Some of the proteins in the present study, namely D1T10 (10+ to 21+) and carbonic anhydrase (15+ to 33+), displayed significantly larger CCSs (5,432 to 8,142 Å 2 ) than those measured in our previous studies. 37,46 The differences in CCS values measured by ion mobility and Orbitrap transient decay are plotted as percent differences (CCS%) in with ion mobility CCSs (CCS% values averaging 5% ± 3%). The CCS values of these protein ions span a large range, 3,500 to 8,000 Å 2 .…”

“…For those experiments, a wide range selected ion monitoring (SIM) method was employed for measurement of Orbitrap CCS. 46 The wide-SIM method involves Orbitrap CCS measurements of 2-4 abundant charge states at the same time. This method allows simultaneous analysis of multiple different ions or charge states but may result in modest overestimation of CCS owing to space charge effects between charge states, as previously described.…”

Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

“…For those experiments, a wide range selected ion monitoring (SIM) method was employed for measurement of Orbitrap CCS. 44 The wide-SIM method involves Orbitrap CCS measurements of 2-4 abundant charge states at the same time. This method allows simultaneous analysis of multiple different ions or charge states but may result in modest overestimation of CCS owing to space charge effects between charge states, as previously described.…”