Bacterial Identification by Protein Mass Mapping Combined with an Experimentally Derived Protein Mass Database

Abstract

A protein mass mapping approach using mass spectrometry (MS) combined with an experimentally derived protein mass database is presented for rapid and effective identification of bacterial species. A prototype mass database from the protein extracts of nine bacterial species has been created by off-line high-performance liquid chromatography (HPLC) matrix-assisted laser desorption/ionization (MALDI) MS, in which the microbiological parameter of bacterial growth time is considered. A numerical method using a statistical weight factor algorithm is devised for matching the protein masses of an unknown bacterial sample against the database. The sum of these weight factors produces a corresponding summed weight factor score for each bacterial species listed in the database, and the database species producing the highest score represents the identity of the respective unknown bacterium. The applicability and reliability of this protein mass mapping approach has been tested with seven bacterial species in a single-blind study by both direct MALDI MS and HPLC electrospray ionization MS methods, and identification results with 100% accuracy are obtained. Our studies have demonstrated that the protein mass database can be rapidly established and readily adopted with relatively less dependency on experimental factors. Furthermore, it is shown that a number of proteins can be detected using a protein sample amount equivalent to an extract of less than 1000 cells, demonstrating that this protein mass mapping approach can potentially be highly sensitive for rapid bacterial identification.

Classification and identification of bacteria using mass spectrometry-based proteomics

“…Recently, Tao and coworkers provided an interesting method for the differentiation of microorganisms [38]. Samples of a bacterial growth culture were analyzed by MALDI-MS between nine and 12 selected times during a growth period between 8 and 48 h. Ten different organisms were used to create a database.…”

“…These include different growth media [19,29,31,[34][35][36][37], growth conditions (e.g., flask vs. test tube [35]; aerated liquid broth vs. solid agar plate growth [14]) and growth time or phase of the organism [19,22,35,38].…”

“…In addition, 2D-PAGE cannot be interfaced directly to MS technologies. Reprinted with permission from [38] © 2004 American Chemical Society.…”

Direct visualisation of peptide hormones in cultured pancreatic islet alpha‐ and beta‐cells by intact‐cell mass spectrometry

“…This technique is now a recognised strategy for the rapid and accurate identification of bacterial species based on intact protein mass databases 4. It enables analysis of low cell numbers, 5, 6 in which core protein profiles remain unchanged by culture media 7. Similar strategies have been applied to reveal peptide profiles from invertebrate neurons, 8, 9 as reviewed by Clynen et al 10.…”

Identification of Pathogens by Mass Spectrometry

“…This methodology has been used successfully for microbial proteomics in the analysis of bacterial species, either pure or in a mixture (60,61 ). Microbial identification has been demonstrated using experimentally derived databases of virus-specific tryptic peptide masses (62 ) and bacterial protein masses (63 ).…”