Invited Paper AS+BI+MI+NS+SA+SS-WeM3
Surface Analysis of Intact Biomolecules: the Bigger They Are the Harder They Fly

Wednesday, November 1, 2017, 8:40 am, Room 13

Secondary ion mass spectrometry (SIMS), as the oldest MSI techniques, gained popularity for analysis of biological samples due to its ability to obtain chemical and spatial information at unmatched lateral resolutions. The use of focused ion beams for desorption and ionization of surface molecules in SIMS affords for this notable spatial resolution over, for example, laser-based MS approaches such as Matrix Assisted Laser Desorption Ionisation (MALDI). However, the excessive energy of the primary ions limits the method to the detection of elements, fragmented molecules and small intact molecular species. This consequently points at the method’s major drawback, which is the difficulty to ionize and detect larger, intact molecular species such as peptides and proteins with great sensitivity. Over the last years, SIMS has been pushing the boundaries by redirecting focus into biomedical applications. Tissue sections and cell imaging has become common practice in research labs all over the world. Now, abundant lipids and small peptides can be studied with different sample surface modifications, where the upper most layer of the surface is sputter coated with a thin layer of metal ((MetA) SIMS – metal –assisted SIMS) or covered with the matrix (ME-SIMS). In both cases the sputtering efficiency and the secondary molecular yield have increased. Here, we studied how ME-SIMS can influence the ionization efficiency of desorbed intact molecules in comparison to MALDI.

First, we imaged mammalian tissue sections that were subjected to a variety of different matrices using a home-built sublimation chamber. Matrix sublimation produces small, homogenous crystal sizes, without the need for solvents that delocalize molecular species. The same or consecutive sections were subsequently analyzed by FTICR-SIMS, to accurately identify the enhanced molecular species of interest specifically intact lipids and metabolites, and by the PHI nano-TOF II for high lateral resolution images and confident identification of said species with tandem MS.Second, de-novo peptide sequencing was performed on endogenous neuropeptides directly from a pituitary gland. Careful sample preparation and the capability of using a 1 Da mass isolation window of the precursor ion followed by a collision-induced dissociation (CID) at 1.5 keV in an activation cell with argon gas enables the molecules to be fragmented in a specific pattern. Neuropeptides up to m/z 2000 were detected and sequenced from the posterior lobe. Further on, we applied it for the characterization of tryptically digested peptides from a variety of tissue sections investigating the applicability to bottom-up proteomics.