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.