International Journal of Mass Spectrometry 312 (2012) 41–44 Contents lists available at ScienceDirect International Journal of Mass Spectrometry j our na l ho me page: www.elsevier.com/locate/ijms Rapid detection of ribosome inactivating protein toxins by mass-spectrometry-based functional assays Miquel D. Antoine, Nathan A. Hagan, Jeffrey S. Lin, Andrew B. Feldman, Plamen A. Demirev ∗ Johns Hopkins University Applied Physics LaboratoryMS21-N454, 11100 Johns Hopkins Rd., Laurel, MD 20723, USA a r t i c l e i n f o Article history: Received 25 April 2011 Received in revised form 5 August 2011 Accepted 10 August 2011 Available online 5 September 2011 This paper is dedicated to Dr. Catherine Fenselau, in recognition of her pioneering role and seminal contributions in the development of biological mass spectrometry and its applications. Keywords: Bio-agent MALDI TOF DNA Bio-threat detection a b s t r a c t A family of structurally related protein toxins extracted from bacteria or plants have a similar mecha- nism of action, namely, blocking protein synthesis by ribosome inactivation. Members of this family of ribosome-inactivating proteins (RIP), classified simultaneously as chemical and biological threat agents, include ricin, abrin, and shiga toxins. We have adapted and streamlined mass spectrometry based func- tional assays, described by others, to develop a rapid, robust, broad-band and fieldable protocol with the primary goal of RIP toxin detection in unknown powder materials. Complementary to intact protein detection and proteomics methods, this protocol involves monitoring the depurination of selected DNA substrates by mass spectrometry. It is being implemented on the CB-TOF system for rapid detection of bio-threats by MALDI MS. Here we illustrate its performance on the example of a plant (ricin) and a bacterial (shiga) RIP toxin. © 2011 Elsevier B.V. All rights reserved. 1. Introduction A family of structurally related and potent cytosolic toxins orig- inating from bacteria or plants have a similar mechanism of action, namely, blocking protein synthesis by ribosome inactivation [1–4]. Members of this family of ribosome-inactivating proteins (RIP) are classified simultaneously as chemical and biological threat agents. They include a number of toxins with varying toxicity (lethal dose) – ricin, abrin shiga and shiga-like toxins (verotoxin), as well as trichosanthin and luffin. Several recent events, widely publicized in the media, have underscored the need for a rapid and reliable method for RIP toxin detection in small sample amounts. Modern biological mass spectrometry is well suited for detecting and char- acterizing all potential bio-threat agents, including protein toxins [5–7]. Recently, new rapid methods for cleanup, concentration and separation have been developed to facilitate confident bio- threat identification by MS in complex backgrounds (soil, sea water, foodstuff, bodily fluids, etc.). For example, with suitable sample preparation protocols intact and/or digested RIP toxin molecules may be extracted and analyzed by MS (both MALDI TOF and ESI MS) [8–21]. Developments have included MS immunoassays, in ∗ Corresponding author. Tel.: +1 443 778 7712. E-mail address: plamen.demirev@jhuapl.edu (P.A. Demirev). which antibodies are first utilized to extract and purify the spe- cific toxin from the mixture. Subsequently, MS analysis reveals the masses of the intact molecules attached to the antibodies. Tandem MS of the intact subunits and/or their tryptic peptides can confi- dently confirm the toxin identification and can also be utilized for characterization of heterogeneities. Such proteomics and antibody- purification protocols, however, may be time consuming, and more difficult to automate and use routinely in a field-laboratory set- ting. Furthermore, the antibodies must be sufficiently specific to capture the respective toxin without cross-reacting with contam- inants. For an unknown sample, multiplexing a MS immunoassay for different RIP toxins would require multiple antibodies with the accompanying increase in cost and logistics. Recently, a number of MS-based functional assays for detec- tion of protein toxins (e.g., botulinum neurotoxins [22,23], anthrax lethal factor [24] and ricin [25–28]) have been described. For exam- ple, the presence of functionally active ricin in a complex sample is inferred by detection of the specific adenine cleavages induced by ricin in the respective substrate molecule (a universally conserved loop in the 28S subunit of eukaryotic rRNA). A CDC-developed pro- tocol incorporates a three-prong approach for ricin detection: toxin immunocapture followed by monitoring the loss of an adenine group in a 12-mer synthetic DNA substrate [26]. Finally, the isolated toxin is identified by traditional bottom-up proteomics approaches. The protocol has been successfully tested for detection of ricin 1387-3806/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijms.2011.08.012