Laserspray Ionization ‐ A New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultra‐High Mass Resolution and Electron Transfer Dissociation Sequencing

A new mass spectrometry (MS) method applicable to proteins which uses laser ablation of a matrix analyte mixture similar to atmospheric pressure (AP) matrix‐assisted laser desorption/ionization (MALDI) but which produces mass spectra with high charge states nearly identical to electrospray ionization (ESI) was recently introduced by us. This new technique called laserspray ionization (LSI) has advantages of speed of analysis and high spatial resolution imaging common with AP‐MALDI and mass range extension on high performance mass spectrometers, as well as improved fragmentation common with multiply charged ESI ions. Because LSI can be interfaced with high performance AP ionization mass spectrometers, it is especially powerful in protein characterization and identification. We have interfaced LSI with a SYNAPT G2 allowing ion mobility spectrometry (IMS) separation of mixtures by charge and cross‐section (size/shape), and in a second dimension, separation with high resolving power by mass‐to‐charge permitting powerful deconvolution of protein mixtures directly from surfaces. Lipids, peptides, and proteins up to ca. 20,000 Da have been observed directly from mouse brain tissue with mass accuracy <5 ppm on an Orbitrap. Sequence analysis has been achieved for peptides and proteins in a single acquisition using an LTQ‐ETD Velos. Myelin basic protein N ‐terminal fragment was identified with a MASCOT score of 71 with the hit confirmed by the mass accuracy of the Orbitrap. The highly charged LSI ions from small proteins produce nearly complete backbone fragmentation as was shown for ubiquitin as well as for labile protein modifications. Further, we show that LSI ions have similar structures as ions produced by ESI as determined by IMS‐MS analysis. We see potential in this technology in high‐throughput proteomics and imaging and are developing methods for characterization of membrane proteins using solvent‐free sample preparation methods.