Next-Generation IMS Instrumentation
Ultra-high Speed and High Mass Resolution IMS
Ultra-high speed image acquisition (>25 pixels/s) permits high spatial resolution imaging from large tissue regions of interest in a practical time frame, driving the application and utility of these technologies. Furthermore, high mass resolution of proteins up to ~20 kDa facilitates identification and drives biological understanding. The combination of these approaches provides a powerful technology for protein analysis .
Figure 1. Analysis of human cystic fibrosis tissue by ultra-high speed MALD-TOF IMS combined with protein identification using high mass resolution MALDI FTICR IMS. Proteomics 11-12: 1678-1689.
In collaboration with Marvin Vestal and his team at SimulTOF Systems, Inc., we are developing TOF/TOF imaging instrumentation and methodologies for high throughput and multiplexed MS/MS imaging experiments (i.e., the selection of multiple precursor/product ion transitions in a single laser shot) using continuous raster sampling [2,3]. This is a significant advancement in new imaging instrumentation:
1. This instrument can acquire data for multiple precursor/product ion transitions in a single laser shot (Figure 2). Previously, a typical MS/MS imaging experiment involved imaging a single selected precursor/product ion transition, producing one image per scan of the tissue.
2. The instrument is capable of a throughput approximately 12-fold faster than a typical MS/MS experiment in an ion trap system. For example, data collected from the sagittal rat brain section shown in Figure 3 was acquired at a spatial resolution of 100 µm in 10 minutes. High throughput analysis is facilitated on this system via a Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz, a high digitizer acquisition rate (up to 50 pixels/second), and continuous laser raster sampling.
Figure 3. High-speed imaging of a sagittal rat brain section. Modified from: J Am Soc Mass Spectrom 22 (6):1022-1031.
Enhanced Ion Transmission Efficiency up to m/z 24 000 for MALDI Protein Imaging Mass Spectrometry
The molecular identification of species of interest is an important part of an imaging mass spectrometry (IMS) experiment. The high resolution accurate mass capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) have recently been shown to facilitate the identification of proteins in matrix-assisted laser desorption/ionization (MALDI) IMS. However, these experiments are typically limited to proteins giving rise to ions of relatively low m/z due to difficulties transmitting and measuring large molecular weight ions of low charge states. Herein we have modified the source gas manifold of a commercial MALDI FT-ICR MS to regulate the gas flow and pressure to maximize the transmission of large m/z protein ions through the ion funnel region of the instrument. By minimizing the contribution of off-axis gas disruption to ion focusing and maximizing the effective potential wall confining the ions through pressure optimization, the signal-to-noise ratios (S/N) of most protein species were improved by roughly 1 order of magnitude compared to normal source conditions. These modifications enabled the detection of protein standards up to m/z 24 000 and the detection of proteins from tissue up to m/z 22 000 with good S/N, roughly doubling the mass range for which high quality protein ion images from rat brain and kidney tissue could be produced. Due to the long time-domain transients (>4 s) required to isotopically resolve high m/z proteins, we have used these data as part of an FT-ICR IMS-microscopy data-driven image fusion workflow to produce estimated protein images with both high mass and high spatial resolutions.
Figure 3. Representative ion images of m/z21,895.717 from rat brain acquired on an FT-ICR MS at a pressure of 0.75 Torr (left) or 2.9 Torr (right). Abstract and graphical abstract: Prentice et al., Enhanced Ion Transmission Efficiency up to m/z 24 000 for MALDI Protein Imaging Mass Spectrometry. Anal Chem, 2018.
1. Spraggins JM, Rizzo DG, Moore JL, Noto MJ, Skaar EP, Caprioli RM (2016) Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis. Proteomics 11-12: 1678-1689. PMCID: PMC5117945
2. Spraggins JM, Caprioli RM (2011) High-speed MALDI-TOF imaging mass spectrometry: rapid ion image acquisition and considerations for next generation instrumentation. J Am Soc Mass Spectrom 22 (6):1022-1031. PMCID: 3514015. Return to text.
3. Prentice BM, Chumbley CW, Caprioli RM (2015) High-speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling. J. Mass Spectrom 50 (4):703-710. PMCID: Not Listed. Return to text.