Experimental\nand Computational Evaluation of Lipidomic\nIn-Source Fragmentation as a Result of Postionization with Matrix-Assisted\nLaser Desorption/Ionization

Matrix-assisted laser desorption/ionization mass spectrometry\nimaging\n(MALDI-MSI) can provide spatially resolved molecular information about\na sample. Recently, a postionization approach (MALDI-2) has been commercially\nintegrated with MALDI-MSI, allowing for bettered sensitivity and consequent\nimproved spatial resolution. While advantages of MALDI-2 have previously\nbeen established, we demonstrate here statistically increased in-source\nfragmentation (ISF) results from postionization with a commercial\ninstrument. Via lipid standard analyses, known MALDI ISF pathways\n(e.g., loss of trimethylamine) were statistically increased in MALDI-2\ncompared to MALDI-1 (65–172% increase in fragmentation). Gas\nphase molecular modeling with density functional theory estimated\nthat the most-weighted virtual orbitals to excite within lipids involve\nester and phosphate bonds. Protonated lipid excitation energies are\nfurthermore red-shifted compared to those of other adduct types [e.g.,\n254 nm for protonated PC(16:0/18:1)] and approach the MALDI-2 laser\nenergy (266 nm). Analysis of rat brain homogenate detected statistically\nmore positive-ion mode peaks with MALDI-2 (1090) than that with MALDI-1\n(719), where Kernel density estimations showed that the majority of\nthis enhancement occurs with low <i>m</i>/<i>z</i> ions (i.e., <i>m</i>/<i>z</i> 75–500).\nTaken together with the lipid standard data, these observations may\nindicate ISF due to postionization. While artifact contributions from\nmatrix blanks were also noted, both experimental and computational\ndata sets suggest that the overall extent of ISF is statistically\nincreased in MALDI-2 compared to MALDI-1.