Detection of norovirus capsid protein in authentic standards and in stool extracts by matrix-assisted laser desorption ionization and nanospray mass spectrometry.

Noroviruses, formerly known as Norwalk-like viruses and small round-structured viruses in the family Caliciviridae, are human pathogens responsible for outbreaks of waterborne, food-borne, person-to-person, hospital, and recreational-water-contact origin (3, 5, 13, 27). Estimated to cause more than 23 million cases of gastroenteritis per year in the United States, norovirus infections are second only to the common cold in disease occurrence (32). The actual number of norovirus cases is unknown, since the disease is not reportable and is difficult to diagnose. Between 1991 and 2000, there was an 11-fold increase in reports of confirmed outbreaks of norovirus (33). In the past 5 years, infections have remained pervasive (32), making virus surveillance a top priority. An added incentive for the development of rapid detection tools is the classification of norovirus as a category B bioterrorism agent by the Centers for Disease Control and Prevention. Among the various potential environmental monitoring matrixes of interest, stool likely represents the most important and arguably the most challenging matrix, due to the abundance and diversity of microorganisms, cell debris, proteins, and lipids contained therein (15). The complexity of stool suggests that one or more purification steps likely are required to extract and enrich the viral fraction prior to analysis, regardless of the type of assay chosen. Methods for accurate measurement of enteric viruses in environmental matrixes remain elusive, aside from those developed for shellfish and large volumes of water (25, 27). One important challenge to the detection of infectious human noroviruses is the limited culturability of this pathogen, which cannot be grown in tissue cultures or hosts other than humans. Detection has traditionally utilized electron microscopy (11). More recent methods include antigen-directed enzyme-linked immunosorbent assays (20) or reverse transcription PCR (RT-PCR) (26, 27). Each of these methods has its own unique challenges, such as target inhibition (PCR), cross-reactivity (enzyme-linked immunosorbent assays), and lengthy sample preparation procedures (electron microscopy), which hinder its effectiveness. As a result, different methods often produce conflicting results (20). The development of a robust, and at the same time rapid, method for virus detection is desirable, because it potentially may allow for a faster and more informed public health response, particularly during outbreak situations. Mass spectrometry (MS) has been proposed as a method for the detection of viruses in a number of settings (6, 28, 35). With recent advances in MS techniques, the ability to detect specific markers in complex samples has been achieved (7). MS typically targets a highly abundant protein in microorganisms of interest (reviewed in reference 1). For viruses, this target is typically a capsid protein (28, 35). Ionization of the selected target proteins frequently is achieved using either matrix-assisted laser desorption ionization (MALDI) or electrospray ionization techniques. In MALDI, the sample is cocrystallized with an energy-absorbing matrix, such as 2,5-dihydroxybenzoic acid, and immobilized on a stainless-steel target plate (1). A laser is then fired upon the crystals, which causes ionization of proteins and peptides. Due to the physics of the interaction, predominantly singly charged ions are produced and ejected (9). MALDI is usually coupled with a time-of-flight (TOF) detector, which aptly measures the time taken for a charged molecule (ion) to travel down a flight tube in a vacuum toward an opposing charge, the travel time being a function of the mass-to-charge ratio (m/z) of the analyte (8). Alternatively, in electrospray ionization MS, the sample is presented in a liquid solvent which is sprayed through a fine-capillary tube that is impinged upon by a powerful high-voltage (≥2-kV) electric field (29). The solvent contained in the resultant charged droplets can be blown off, leaving behind ions which are then electromagnetically focused and detected by MS. In contrast to MALDI, electrospray ionization produces predominantly multiply charged ions (34). The sensitivity of this assay can be improved by using nanospray sample introduction techniques. Both MALDI and electrospray ionization are effective tools for the ionization of peptides for mass spectrometric analysis. Comparative studies have demonstrated the pros and cons of both methods (22). The present study employed MALDI-TOF and electrospray ionization MS (i) to identify biomarkers for norovirus, (ii) to demonstrate the possibility of norovirus detection using three different proteomic MS approaches, (iii) to determine the limit of detection of MALDI-TOF MS using virus-like particles (VLPs) of defined purity and quantity as a surrogate for the actual infective target, (iv) to evaluate a rapid protocol for the efficient purification of virus particles from human stool, and (v) to explore the potential of MS for accurate detection of biomarkers for norovirus in a complex sample matrix, i.e., in fortified extracts of clinical stool samples.