Laserspray Ionization, a New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation

Abstract

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

“…A nanoESI source was used on a Waters SYNAPT G2 mass spectrometer instrument to perform AP-LSI as previously described [49,50]. Scan time was set at 1 scan per s and up to 1 min acquisition time.…”

Localization and imaging of gangliosides in mouse brain tissue sections by laserspray ionization inlet

“…I-C) ( 52,(64)(65)(66). This technique offers further improvements to spatial resolution and high-throughput acquisitions ( 65,66 ), as only a single laser shot is required to obtain a mass spectrum. This is especially important for imaging mass spectrometry, in which LSII has recently been developed by Richards et al ( 65 ) to image phospholipids and sulfatides from mouse brain tissue using solvent-free sample preparation, inlet ionization (supplementary Fig.…”

“…Additionally, LSII often employs transmission geometry (TG) in which the laser is aligned 180° to the sample/orifi ce and ablates the analyte from the backside (supplementary Fig. I-C) ( 52,(64)(65)(66). This technique offers further improvements to spatial resolution and high-throughput acquisitions ( 65,66 ), as only a single laser shot is required to obtain a mass spectrum.…”

“…2,5-DHAP was used as matrix and applied on top of the tissue using the spray coating approach ( 86 ). Improved results were obtained by combining spray coating with the precoated approach ( 64,66,87 ), in which the matrix was also placed below the tissue. The enhanced ionization is rationalized by the extraction of lipids into the matrix from both surfaces of the 10 µm thick tissue section.…”

“…I-D) of 0.7 ng of protein digest on column ( 91 ). We further hypothesize that shorter acquisition times will simplify convoluted mass spectral data relative to sample complexity and charge state distributions associated with the direct analysis of tissue material ( 66 ) and that deconvolution programs will assist in the interpretation of LSII ions similar to those used in ESI ( 92 ). Besides addressing pressing needs for enhanced desolvation of the matrix in inlet ionization through instrument modifications, current research aimed at imaging gangliosides includes the search for improved matrix materials for ease in desolvation/ionization at lower inlet temperatures; improving sample preparation techniques, particularly matrix preparation and application, to visualize lower abundance gangliosides such as GT1 and GQ1 from tissue while enhancing sensitivity and spatial resolution; shortening analyses time (<10 min for one mouse brain tissue section) ( 57 ); and enhancing dynamic range by expanding applications to LSII-ion mobility spectrometry-MS ( 54,55,59,68 ) and MS n imaging for the differentiation of ganglioside isomers.…”