Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

Abstract

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.

A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics

“…This is particularly true in those cases where the protein sequence database contains many homologous proteins and splice isoforms (e.g. in the analysis of higher eukaryotes), or when the database intentionally includes sequences from multiple organisms [245]. …”

Rapid Characterization of Microorganisms by Mass Spectrometry—What Can Be Learned and How?

“…Most of the literature discussing automatable rapid analysis of unfractionated microorganisms deals with bacteria and bacterial spores [1][2][3][4][5][6][7][8][9], for example B. anthracis and other species defined as biological warfare agents. Fungi, including yeast, are susceptible to the same strategies [10][11][12]. Viruses provide a challenging current frontier for unfractionated characterization, since they are hard to lyse, synthesize a smaller selection of characteristic proteins, and are more difficult to culture.…”

Preview: A Program for Surveying Shotgun Proteomics Tandem Mass Spectrometry Data

“…In some cases, the loss of sensitivity can be catastrophic, for example 90% loss of sensitivity at the protein level due to non-specific digestion in a sample of the fungus Fusarium graminearum 30 . By providing a complete statistical overview of the data set before setting parameters for lengthy database searches, Preview can help improve both sample preparation and data analysis, ensuring that proteomics experiments yield maximum biological information.…”