Investigative proteomics: Identification of an unknown plant virus from infected plants using mass spectrometry

Abstract

We describe the identification of a previously uncharacterized plant virus that is capable of infecting Nicotiana spp. and Arabidopsis thaliana. Protein extracts were first prepared from leaf tissue of uninfected tobacco plants, and the proteins were visualized with two-dimensional electrophoresis (2-DE). Matching gels were then run using protein extracts of a tobacco plant infected with tobacco mosaic virus (TMV). After visual comparison, the proteins spots that were differentially expressed in infected plant tissues were cut from the gels and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Tandem mass spectrometry data of individual peptides was searched with SEQUEST. Using this approach we demonstrated a successful proof-of-concept experiment by identifying TMV proteins present in the total protein extract. The same procedure was then applied to tobacco plants infected with a laboratory viral isolate of unknown identity. Several of the differentially expressed protein spots were identified as proteins of potato virus X (PVX), thus successfully identifying the causative agent of the uncharacterized viral infection. We believe this demonstrates that HPLC-MS/MS can be used to successfully characterize unknown viruses in infected plants. (J Am Soc Mass Spectrom 2003, 14, 736 -741)

La Autoantigen Mediates Oxidant Induced De Novo Nrf2 Protein Translation

“…Parent peptide mass error tolerance was set at 2.0 amu and fragment ion mass tolerance was set at 1.0 amu during the search. The criteria used for a preliminary positive peptide identification are the same as previously described, namely a dCn score Ͼ 0.08, and Xcorr Ͼ1.8 for ϩ1, Xcorr Ͼ2.5 for ϩ2, and Xcorr Ͼ3.5 for ϩ3 charged peptide precursor ions (16). Peptides matching proteins of interest were confirmed by visual examination of the spectra.…”

Genomic and proteomic analysis of the effects of cannabinoids on normal human astrocytes

“…In-gel digestion and nano high performance liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS) were performed as described (Breci et al, 2005). Briefly, gel slices were destained (Breci et al, 2005;Cooper et al, 2003) and digested with trypsin (Wilm et al, 1996). The tryptic peptides were extracted with 5% formic acid/50% CH 3 CN.…”

Identification of plant viruses using one-dimensional gel electrophoresis and peptide mass fingerprints

“…Traditional diagnostic methods of symptom evaluation, vector specificity, host range studies, and electron microscopy to visualize particle and inclusion size and shape tend to be more generic than molecular methods. Often, two or more assays are combined as part of the diagnosis process (Cooper et al, 2003).…”

“…By using an appropriate scoring algorithm, the closest match or matches are identified. Cooper et al (2003) identified Tobacco mosaic virus and Potato virus X using two-dimensional SDS PAGE to compare proteomes of infected and Page 6 of 18 A c c e p t e d M a n u s c r i p t uninfected plant tissues. Differential spots were excised, digested by trypsin, analyzed by MALDI-TOF MS and PMFs generated.…”

“…In this assay the virus was identified from an extract of total plant proteins, and it does not require lengthy or complicated purification of virus particles from host proteins (eg Wang et al, 2007;Blouin et al, 2009). It does not require two-dimensional M a n u s c r i p t separation by SDS PAGE (eg Cooper et al, 2003) and it utilises a rapid in-gel trypsin digestion procedure. Further, single MS analysis rather than tandem MS (eg Blouin et al, 2009) was sufficient for identifying the viruses tested.…”