LESA MS Imaging of Heat-Preserved and Frozen Tissue: Benefits of Multistep Static FAIMS

Abstract

We have previously demonstrated liquid extraction surface analysis (LESA) high field asymmetric waveform ion mobility spectrometry (FAIMS) mass spectrometry imaging of proteins in thin tissue sections of brain and liver. Here, we present an improved approach which makes use of multiple static FAIMS parameters at each sampled location and allows a significant improvement in the number of proteins, lipids and drugs that can be imaged simultaneously. The approach is applied to the mass spectrometry imaging of control and cassette-dosed rat kidneys. Mass spectrometry imaging of kidneys typically requires washing to remove excess hemoglobin; however, that is not necessary with this approach. Multi-step static FAIMS mass spectrometry resulted in a six-to sixteen-fold increase in the number of proteins detected in the absence of FAIMS, in addition to smaller increases over single step static FAIMS (chosen for optimum transmission of total protein ions). The benefits of multi-step static FAIMS mass spectrometry for protein detection are also shown for sections of testes. The numbers of proteins detected following multistep FAIMS increased between two-and threefold over single step FAIMS, and between two-and fourteen-fold over LESA alone. Finally, to date, LESA mass spectrometry of proteins in tissue has been undertaken solely on fresh frozen samples. In this work, we demonstrate that heat-preserved tissues are also suitable for these analyses. Heat preservation of tissue improved the number of proteins detected by LESA MS for both kidney and testes tissue (by between two-and fourfold). For both tissue types, the majority of the proteins additionally detected in the heat-treated samples were subsequently detected in the frozen samples when FAIMS was incorporated. Improvements in the numbers of proteins detected were observed for LESA FAIMS MS for the kidney tissue; for testes tissue fewer total proteins were detected following heat preservation, however approximately one third were unique to the heat preserved samples.

A Critical and Concise Review of Mass Spectrometry Applied to Imaging in Drug Discovery

“…For example, liquid extraction surface analysis (LESA) has been demonstrated to add extra sensitivity at the expense of spatial resolution [18][19][20] when applied to metabolic profiling, 21 drug imaging, 3 or challenging intact protein targets. 22,23 Combining similar surface droplet microjunction techniques with pre-ionization chromatography increases the specificity and range of detected targets, but compromises on overall throughput. 24 There are extensive other ionization methods that readers could continue to consider for future roles in MSI in R&D, including variations on the themes just discussed [such as laser ablation electrospray ionization 25 (LAESI), laser desorption ionization 26 (LDI), or NanoDESI 27 ] or more tangential techniques such as pixel imaging mass spectrometry.…”

“…For example, liquid extraction surface analysis (LESA) has been demonstrated to add extra sensitivity at the expense of spatial resolution 18–20 when applied to metabolic profiling, 21 drug imaging, 3 or challenging intact protein targets. 22,23 Combining similar surface droplet microjunction techniques with pre-ionization chromatography increases the specificity and range of detected targets, but compromises on overall throughput. 24…”

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

“…Smaller proteins are often reported; analysis of mouse tissues resulted in the detection of proteins up to ∼20 kDa, including calmodulin (16.8 kDa) and α-globin (15.0 kDa) identified by LESA tandem mass spectrometry (see the section below) [30]. Different proteins may be detected based on tissue storage protocol, as evidenced by the analysis of heat-treated versus fresh frozen tissue [31].…”

“…To address that challenge, LESA has been coupled to high-field asymmetric waveform ion mobility spectrometry (FAIMS) [32]. FAIMS can improve the signal-to-noise ratio for protein ion signals by filtering out interfering signals arising from other extracted analytes from both tissue [30,31,33] and bacterial colonies [33,34]. Travelling wave ion mobility spectrometry (TWIMS) [35] has been combined with LESA MS [36] to determine the 3D shape of protein ions (in the form of collision cross sections) but its utility as a FAIMS-like filter has yet to be explored.…”

In situ analysis of intact proteins by ion mobility mass spectrometry

“…This capability allows for greater flexibility in the design of a FAIMS experiment, e.g., the inclusion of multiple FAIMS settings, thereby transmission of different ions, in a single acquisition. Using a multistep static FAIMS approach in the LESA MSI of sections of rat kidney and rat testes, it was possible to increase the number of proteins detected several fold over a single step FAIMS approach [36]. For example, 55 proteins were detected in a multistep FAIMS analysis of fresh frozen rat kidney tissue compared with seven proteins detected in a single-step analysis.…”