A high resolution atmospheric pressure matrix-assisted laser desorption/ionization-quadrupole-orbitrap MS platform enables in situ analysis of biomolecules by multi-mode ionization and acquisition

Abstract

Introduced in 2000, atmospheric pressure (AP)/matrix-assisted laser desorption/ionization (MALDI) has attracted substantial attention in the mass spectrometry community due to its ease of sample introduction and handling, interchangeability with ESI source and capability of analyzing volatile species. In this study, an AP/MALDI source with ultra-high spatial resolution was coupled to a Q Exactive HF orbitrap mass spectrometer for high resolution in situ analysis by MALDI, laserspray ionization (LSI) and matrix assisted ionization (MAI) without instrument modification. LSI and MAI generated multiply charged ions, which expanded the mass detection range and improved fragmentation efficiency. Full MS, targeted MS/MS, data dependent acquisition (DDA) and parallel reaction monitoring (PRM) acquisitions were performed on peptide and protein standards, tissue extracts and tissue sections for in depth characterization of various biomolecules. High resolution full MS and MS/MS images were obtained from crustacean and rat tissues with pixel size less than 30 μm. Overall, AP/MALDI-Q-Orbitrap is a fast scanning instrument that is capable of performing multiple types of ionization and multiple acquisition modes without instrument modification. This instrument platform provides an attractive alternative to other high resolution MALDI instruments.

Keywords: Atmospheric pressure ionization; High resolution accurate mass; Laserspray ionization; Mass spectrometry imaging; Matrix-assisted laser desorption/ionization; Neuropeptide.

Figure 1. Comparisons of full MS spectra of MALDI, LSI and MAI MS analysis of bradykinin (1059 Da) (a, b & c) and neuropeptide Y (4269 Da) (d, e & f)

Singly charged bradykinin peak was observed under MALDI (a), LSI (b) and MAI (c) conditions, while doubly charged bradykinin peak could only be observed under LSI (b) and MAI (c) conditions. No neuropeptide Y related peak could be detected at MALDI condition when CHCA was used as matrix (d). Singly and doubly charged ions were detected at LSI condition (e) and +2 to +7 charged neuropeptide Y peaks were detected under MAI condition (f). All peaks were detected with low ppm mass errors as labeled.

Figure 2. Comparison of MS/MS spectra of bradykinin ionized by MALDI, LSI and MAI

(a) MS/MS of singly charged bradykinin under MALDI condition at NCE 33. (b) MS/MS of singly charged bradykinin under LSI condition at NCE 33. (c) MS/MS of doubly charged bradykinin under LSI condition at NCE 32. (d) MS/MS of singly charged bradykinin under MAI condition at NCE 33. (e) MS/MS of doubly charged bradykinin under MAI condition at NCE 32. All MS/MS spectra were acquired with isolation window of 2 m/z and resolution of 30,000 at m/z 200. Y-axis represents relative signal abundance.

Figure 3. Full MS and MS/MS analyses of crustacean PO and rat pituitary gland neuropeptide extractions

(a) MALDI full MS spectrum of rock crab PO neuropeptide extraction at low m/z region (m/z 620–1000). (b) MALDI full MS spectrum of rock crab PO neuropeptide extract at normal m/z region (m/z 1000–2000). (c) LSI full MS and MS/MS spectra of pituitary neuropeptide extract. Neuropeptides were tentatively identified by accurate mass matching within 10 ppm of theoretical masses. Targeted MS/MS were performed on selected peaks to verify the identifications. NCEs were optimized for each ion.

Figure 4. High resolution MSI of crustacean brain tissue section with 30 μm pixel size

(a) Optical image of brain tissue section. (b–q) Selected ion images of crustacean neuropeptides in the following families: RFamide (b, e, f, i, j & q), tachykinin (c, l & n) and allatostatin A (d, g, h, k, m & p). Neuropeptide identifications were tentatively performed by accurate mass matching against the in-house constructed crustacean neuropeptide database with a mass tolerance of 10 ppm.

Figure 5. High resolution MSI of rat cerebellum section with 25 μm pixel size

(a) Microscopic optical image of cresyl violet stained rat cerebellum after MSI acquisition. The laser burnt mark could be observed on the zoomed in optical. The distance between each line is 25 μm. (b) Selected ion images that have distributions all around the tissue. (c) Selected ion images that have distribution only in the center of cerebellum. (d) Selected ion images that have distribution at the peripheral area of cerebellum. (e) Selected ion images that have special distribution. Identifications were tentatively assigned by database search with a mass tolerance of 10 ppm in METLIN. All MSIs were normalized to TIC and presented without smoothing.