Detection of Norovirus Capsid Protein in Authentic Standards and in Stool Extracts by Matrix-Assisted Laser Desorption Ionization and Nanospray Mass Spectrometry

Abstract

Mass spectrometry (MS) represents a rapid technique for the identification of microbial monocultures, and its adaptation to the detection of pathogens in real-world samples is a public health and homeland security priority. Norovirus, a leading cause of gastroenteritis in the world, is difficult to monitor because it cannot be cultured outside the human body. The detection of norovirus capsid protein was explored using three common MS-based methods: scanning of intact proteins, peptide mass fingerprinting, and peptide sequencing. Detection of intact target protein was limited by poor selectivity and sensitivity. Detection of up to 16 target peptides by peptide mass fingerprinting allowed for the reproducible and confident (P < 0.05) detection of the 56-kDa norovirus capsid protein in the range of 0.1 ؋ 10 ؊12 to 50 ؋ 10 ؊12 mol in authentic standards of recombinant norovirus virus-like particles (VLPs). To explore assay performance in complex matrixes, a non-gel-based, rapid method (2 to 3 h) for virus extraction from human stool was evaluated (72% ؎ 12% recovery), and additional analyses were performed on norovirus-free stool extracts fortified with VLPs. Whereas peptide mass fingerprinting was rendered impractical by sample interferences, peptide sequencing using nanospray tandem MS facilitated unambiguous identification of >250 fmol of capsid protein in stool extracts. This is the first report on MS-based detection of norovirus, accomplished by using structurally identical, noninfective VLPs at clinically relevant concentrations. It represents an important milestone in the development of assays for surveillance of this category B bioterrorism agent.

Norovirus diagnostics: options, applications and interpretations

“…Detection of no rovirus through ma ss spectrometry has been reported using feces spiked with recombinant no rovirus vi rus-like particles, but requiring a 2-3 h physical fractionation step [82]. The utility of this method for the detection of no roviruses in clinical samples with varying concentrations of virus particles and diverse virus genotypes has not been evaluated to date.…”

Nanotechnology in proteomics

“…Examples of current applications include large-scale metabolomics investigations [19], studies of enzyme and their activity [20,21], and protein discovery and characterization [16]. Translational applications of NSI-MS outside the domain of proteomics include microbiology investigations to identify novel bacterial [22] and viral [23] proteins, pharmacokinetic research [24], drug discovery [24,25], and quality-control applications [26].…”

Breast cancer-specific serum peptide profiles

“…MS now permits the display of hundreds of small-to medium-sized peptides with only microliters of serum. Several recent reports have advocated the use of the MS approach to determine specific patterns that are indicative of ovary, pancreas, prostate, bladder, colon and breast cancers [5][6][7][8][9][10][11][12][13][14][15][16] . The majority of these studies have used low resolution instruments and surface-enhanced laser desorption/ionization (SELDI) [5][6][7][8][9][10][11]17,18] .…”

“…The majority of these studies have used low resolution instruments and surface-enhanced laser desorption/ionization (SELDI) [5][6][7][8][9][10][11]17,18] . Several recent studies have been performed on high resolution instrument with a matrix-assisted laser desorption/ionization (MALDI) ion source and a time-of-flight (TOF) ion detector [12][13][14][15][16]19,20] . The more sophisticated forms of the instrument, such as the fourier transfer (FT) MS/MS, allow the identification of peptide sequences [12] .…”