Experiments were performed to examine the feasibility of MS depth profiling of animal tissue by ~75 fs, 800 nm laser pulses to expose underlying layers of tissue for subsequent MS analysis. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to analyze phospholipids and proteins from both intact bovine eye lens tissue and tissue ablated by ultrashort laser pulses. Laser desorption postionization (LDPI-MS) with 10.5 eV single photon ionization was also used to analyze cholesterol and other small molecules in the tissue before and after laser ablation. Scanning electron microscopy was applied to examine the ablation patterns in the tissue and estimate the depth of the ablation craters. Ultrashort pulse laser ablation was found able to remove a layer of several tens of micrometers from the surface of eye lens tissue while leaving the underlying tissue relatively undamaged for subsequent MS analysis. MS analysis of cholesterol, phospholipids, peptides, and various unidentified species did not reveal any chemical damage caused by ultrashort pulse laser ablation for analytes smaller than ~6 kDa. However, a drop in intensity of larger protein ions was detected by MALDI-MS following laser ablation. An additional advantage was that ablated tissue displayed up to an order of magnitude higher signal intensities than intact tissue when subsequently analyzed by MS. These results support the use of ultrashort pulse laser ablation in combination with MS analysis to permit depth profiling of animal tissue.

The small molecular analyte 3,5-dibromotyrosine (Br2Y) and chitosan-alginate polyelectrolyte multilayers (PEM) with and without adsorbed Br2Y were analyzed by laser desorption postionization mass spectrometry (LDPI-MS). LDPI-MS using 7.87 eV laser and tunable 8 – 12.5 eV synchrotron vacuum ultraviolet (VUV) radiation found that desorption of clusters from Br2Y films allowed detection by ≤8 eV single photon ionization. Thermal desorption and electronic structure calculations determined the ionization energy of Br2Y to be ~8.3±0.1 eV and further indicated that the lower ionization energies of clusters permitted their detection at ≤8 eV photon energies. However, single photon ionization could only detect Br2Y adsorbed within PEMs when using either higher photon energies or matrix addition to the sample. All samples were also analyzed by 25 keV Bi3+ secondary ion mass spectrometry (SIMS), with the negative ion spectra showing strong parent ion signal which complemented that observed by LDPI-MS. However, the negative ion SIMS appeared strongly dependent on the high electron affinity of this specific analyte and the analyte’s condensed phase environment.

The heptapeptide ARHPHPH was identified from biofilms and planktonic cultures of two different strains of Enterococcus faecalis, V583 and ATCC 29212, using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). ARHPHPH was also imaged at the boundary of cocultured, adjacent E. faecalis and Escherichia coli (ATCC 25922) biofilms, appearing only on the E. faecalis side. ARHPHPH was proteolyzed from κ-casein, a component in the growth media, by E. faecalis microbes. Additionally, top down and bottom up proteomic approaches were combined to identify and spatially locate multiple proteins within intact E. faecalis V583 biofilms by MALDI-MS. The resultant tandem MS data were searched against the NCBInr E. faecalis V583 database to identify thirteen cytosolic and membrane proteins which have functional association with the cell surface. Two of these proteins, enolase and GAPDH, are glycolytic enzymes known to display multiple functions in bacterial virulence in related bacterial strains. This work illustrates a powerful approach for discovering and localizing multiple peptides and proteins within intact biofilms.

The potential of laser desorption postionization mass spectrometry (LDPI-MS) imaging for small molecule quantification is demonstrated here. The N-methylpiperazine acetamide of (MPA) ampicillin was adsorbed into polyelectrolyte multilayer surface coatings composed of chitosan and alginate, both high molecular weight biopolymers. These MPA-ampicillin spiked multilayers were then shown to inhibit the growth of E. faecalis biofilms that play a role in early stage infection of implanted medical devices. Finally, LDPI-MS imaging using 7.87 eV single photon ionization was found to detect MPA-ampicillin with the multilayers before and after biofilm growth with limits of quantification and detection of 0.6 and 0.3 nmoles, respectively. The capabilities of LDPI-MS imaging for small molecule quantification are compared to those of MALDI-MS. Furthermore, these results indicate that 7.87 eV LDPI-MS imaging should be applicable to quantification of a range of small molecular species on a variety of complex organic and biological surfaces. Finally, while MS imaging for quantification was demonstrated here using LDPI, it is a generally useful strategy that can be applied to other methods.