Constructing a Tandem Mass Spectral Library for Forensic Ricin Identification.

Ricin, a protein found in castor seeds, is a lethal toxin that is designated as a category 2 select agent, and cases of attempted ricin poisoning are relatively common. Many methods to detect protein toxins such as ricin use targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify toxin peptides, usually tryptic peptides. The successful use of untargeted methods has also been reported. However, the use of untargeted proteomics methods, including database search, for peptide and protein identification is less common in forensic practice and may be unfamiliar to forensic science practitioners. Here, we propose a method to create spectral libraries of tryptic ricin peptides and use these libraries for ricin identification by spectral library search, which may be more familiar to forensic scientists because of the use of spectral libraries in small molecule identification. Peptide spectral libraries offer a direct comparison to an authentic standard, a key element of forensic analysis, but have not previously been used in a forensic context. To construct these spectral libraries, two pure ricin samples (one from a proposed standard reference material) were digested with trypsin and analyzed using a standard shotgun LC-MS/MS protocol. Spectral libraries were created from resulting tryptic peptides identified from filtered search results from four database search tools. The library was then used in a search using SpectraST on forensically realistic castor seed extracts. These castor seed samples were made using the crude methods commonly encountered in real-world ricin cases. Analysis showed that the spectral library search resulted in more peptides identified from crude castor seed samples compared to MS-GF+ and Sequest plus Percolator database searches. These results, the first published use of spectral library search to detect protein toxins in forensically relevant samples, suggest that computational comparison of putative ricin peptide spectra to library spectra can be an effective method to detect ricin in an unknown sample. Data are available via ProteomeXchange with identifier PXD013711.

Two-dimensional and three-dimensional dynamic imaging of live biofilms in a microchannel by time-of-flight secondary ion mass spectrometry.

A vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface), was employed for in situ chemical imaging of live biofilms using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Depth profiling by sputtering materials in sequential layers resulted in live biofilm spatial chemical mapping. Two-dimensional (2D) images were reconstructed to report the first three-dimensional images of hydrated biofilm elucidating spatial and chemical heterogeneity. 2D image principal component analysis was conducted among biofilms at different locations in the microchannel. Our approach directly visualized spatial and chemical heterogeneity within the living biofilm by dynamic liquid ToF-SIMS.

In situ molecular imaging of a hydrated biofilm in a microfluidic reactor by ToF-SIMS.

A novel microfluidic reactor for biofilm growth and in situ characterization using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was constructed to enable two-dimensional chemical imaging of hydrated biofilms. We demonstrate the detection of characteristic fatty acid fragments from microfluidic reactor-grown biofilms and illustrate advantages of hydrated-state ToF-SIMS imaging.