Ultra-High Mass Resolving Power, Mass Accuracy, and Dynamic Range MALDI Mass Spectrometry Imaging by 21-T FT-ICR MS

Abstract

Detailed characterization of complex biological surfaces by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) requires instrumentation that is capable of high mass resolving power, mass accuracy, and dynamic range. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers the highest mass spectral performance for MALDI MSI experiments, and often reveals molecular features that are unresolved on lower performance instrumentation. Higher magnetic field strength improves all performance characteristics of FT-ICR; mass resolving power improves linearly, while mass accuracy and dynamic range improve quadratically with magnetic field strength. Here, MALDI MSI at 21T is demonstrated for the first time: mass resolving power in excess of 1 600 000 (at m/z 400), root-mean-square mass measurement accuracy below 100 ppb, and dynamic range per pixel over 500:1 were obtained from the direct analysis of biological tissue sections. Molecular features with m/z differences as small as 1.79 mDa were resolved and identified with high mass accuracy. These features allow for the separation and identification of lipids to the underlying structures of tissues. The unique molecular detail, accuracy, sensitivity, and dynamic range combined in a 21T MALDI FT-ICR MSI experiment enable researchers to visualize molecular structures in complex tissues that have remained hidden until now. The instrument described allows for future innovative, such as high-end studies to unravel the complexity of biological, geological, and engineered organic material surfaces with an unsurpassed detail.

Figure 1

Mass resolution and sensitivity improve with longer transient length. Within a 100 mDa mass range, seven different peaks are detected, which belong to six different lipid species. Of these, five are unresolved at 0.77 s. While distinguishable at 1.55 s, all seven peaks are fully resolved only at 3.1 s transient. These seven peaks correspond to the isotopologues of the monoisotopic species, typically the ¹³C ion, as in (a), (b), (f), and (g). Other species are also present, corresponding to the ¹³C₃ isotopologue, as in (d) and (e). The ¹⁸O¹³C isotopologue of [PC(34:1)+Na]⁺ is also resolved (c) from the ¹³C₃ isotopologue of the same parent species.

Figure 2

Representative images of close mass differences in negative and positive mode, from a single, scan. Images are total ion current normalized. Positive mode lipid spectra have a significant number of mass differences of 2.4 mDa (a), representing the difference between ¹²C₂ and ²³Na¹H. 2.4 mDa differences are baseline resolved, and show significantly different distributions within brain tissue (b and c). There are nearly 200 such differences in the averaged spectra, shown in 0.5 mDa bins (d). Similarly, negative mode spectra have 1.79 mDa mass differences (e). These 1.79 mDa differences are resolved to better than full-width half-maximum, differentiated well enough to distinguish them in brain tissue (f and g). The of 1.79 mDa mass difference is relatively uncommon in negative mode, but mass differences of 10 mDa or less occur approximately 500 times in the averaged spectra, shown in 0.25 mDa bins (h).

Figure 3

Single on-tissue mass spectrum illustrates high dynamic range per pixel. Peaks were picked at a threshold of six standard deviations above the baseline noise. Dynamic range in a single average pixel of at 536:1 is demonstrated here at pixel number 10 000, (a). Mass scale expanded segment around most abundant peak [PC 34:1 + K]⁺ (b). Further, peak at 798.5410 generates a bright image (b). One of the lowest S/N peaks, the ¹³C₂ isotope of [PE 46:5 + H]⁺ (c) while less clear, still yields informative molecular images, being highlighted especially in the ventricles (images are TIC normalized).

Figure 4

Error histogram and average mass error of tentatively identified lipids after internal calibration. Measured mass error histogram of 139 phosphatidylcholine lipids; the rms error is 61.12 ppb. Bin size = 10 ppb. (a), Lipid identifications by class. A tolerance of ±250 ppb results in 702 potential lipids identified within 150 ppb of their expected mass (b).

Figure 5

Relative abundance of identified lipids by cation and anion for selected classes. In the positive mode, the three major cations (proton, sodium, and potassium) are aligned next to one another, showing the same relative percentages between species, from the most abundant species (PC 34:1) and the other PCs above 3% (a), as well as for the lower abundant species down to the least abundant species with all three cations represented, PC 44:12 (b). The relative ionization rate between K⁺, H⁺, and Na⁺ hold strictly true down to 1.5%, and generally true down to 0.05%. While the dynamic range is lower for negative mode, we see many potential identifications for many lipid classes (c). We further observe a similar ability to identify potential lipids as low as 0.15% of the most abundant peak (PI 38:4), for a range of nearly three orders of magnitude from the summed spectra (d).