We present an approach that speeds up protein solid-state NMR (SSNMR) by 5–20 fold by using paramagnetic doping to condense data-collection time (to ~0.2 s/scan), overcoming a long-standing limitation on slow recycling due to intrinsic 1H T1 longitudinal spin relaxation. By employing low-power schemes under magic-angle spinning at 40 kHz, we show that two-dimensional 13C/13C and 13C/15N SSNMR spectra can be attained for several to tens of nano-moles of β-amyloid fibrils and ubiquitin in just 1–2 days.

Covering: 2013 The ability to transfer molecular species to the gas phase and ionize them is central to the study of natural products and other molecular species by mass spectrometry (MS). MS-based strategies in natural products have focused on a few established ion sources, such as electron impact and electrospray ionization. However, a variety of other ion sources are either currently in use to evaluate natural products or show significant future promise. This review discusses these various ion sources in the context of other articles in this special issue, but is also applicable to other fields of analysis, including materials science. Ion sources are grouped based on the current understanding of their predominant ion formation mechanisms. This broad overview groups ion sources into the following categories: electron ionization and single photon ionization; chemical ionization-like and plasma-based; electrospray ionization; and, laser desorption-based. Laser desorption-based methods are emphasized with specific examples given for laser desorption postionization sources and their use in the analysis of intact microbial biofilms. Brief consideration is given to the choice of ion source for various sample types and analyses, including MS imaging.

Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes, then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at ~100 μm spatial resolution. Spatial resolution of ~20 μm was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single photon ionization source.

Mass spectrometry (MS) imaging is increasingly being applied to probe the interfaces of biomaterials with invasive microbial biofilms, human tissue, or other biological materials. Laser desorption vacuum ultraviolet postionization with ∼75 fs, 800 nm laser pulses (fs-LDPI-MS) was used to collect MS images of a yeast-Escherichia coli co-culture biofilm. The method was also used to depth profile a three-dimensionally structured, multispecies biofilm. Finally, fs-LDPI-MS analyses of yeast biofilms grown under different conditions were compared with LDPI-MS using ultraviolet, nanosecond pulse length laser desorption as well as with fs laser desorption ionization without postionization. Preliminary implications for the use of fs-LDPI-MS for the analysis of biomaterials interfaces are discussed and contrasted with established methods in MS imaging.

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.