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Characterization of Microorganisms by MALDI Mass Spectrometry Catherine E. Petersen, Nancy B. Valentine, and Karen L. Wahl Summary
Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for characterization and analysis of microorganisms, specifically bacteria, is described here as a rapid screening tool. The objective of this technique is not comprehensive protein analysis of a microorganism but rather a rapid screening of the organism and the accessible protein pattern for characterization and distinction. This method is based on the ionization of the readily accessible and easily ionizable portion of the protein profile of an organism that is often characteristic of different bacterial species. The utility of this screening approach is yet to reach its full potential but could be applied to food safety, disease outbreak monitoring in hospitals, culture stock integrity and verification, microbial forensics, or homeland security applications.
Key words: Bacteria, Spores, Microorganisms, Mass spectrometry, MALDI, Proteins, Fingerprinting.
There is an ever-increasing need for the consistent and rapid identification of intact microorganisms. Mass spectrometry is a powerful analytical tool that can also be used for screening microorganisms rapidly. Matrix-assisted laser desorption/ioniza-tion-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify microorganisms based on expressed protein profiles. MALDI-TOF MS provides rapid analysis time (< 1 min/ sample analysis), has low sample volume requirements (<1 11L of fluid), and yields very specific and unbiased analysis based on the molecular weights of true components of the sample. The high sensitivity and high tolerance toward contaminants have made MALDI-TOF MS a viable technique for the analysis of complex biological samples.
MALDI-TOF MS has established itself as an analytical technique having the ability to identify bacteria to the species level in pure cultures and simple mixtures of bacteria (1). It can be used as a rapid screening tool in distinguishing between pathogenic and nonpathogenic species, which potentially makes it a powerful tool in countering terrorism. MALDI-TOF MS has also been successfully extended to the identification of sporulated varieties of Gram-positive strains of Bacillus with modification to the sample preparation techniques (2).
The screening of bacterial samples by mass spectrometry for quick identification can be accomplished with direct analysis of a subset of the protein profile by MALDI-TOF MS analysis. While MALDI-TOF MS is known to have a wide dynamic range of analysis it is also a competitive ionization method and therefore may not yield a profile representative of all components present in the sample. The chemical complexity in most vegetative bacterial samples along with the many orders of magnitude difference in concentration often results in only a minor number of cell components observed by direct MALDI-TOF MS analysis of the entire cell. However, this only presents a significant challenge if the desired result is the complete profiling of the cell contents. A reproducible pattern of putative proteins is readily observed from simple sample preparation and analysis of bacterial cultures (3, 4). The sample preparation and analysis method described here was developed as a rapid screening tool for identification of microorganisms without individual protein identification (5, 6). While relatively straightforward, there are a few steps that are helpful to follow in order to successfully obtain data from microorganisms directly by MALDI-TOF MS. The purpose of this chapter is to provide some guidance on one possible approach to analysis of microorganisms by MALDI-TOF MS. This is not the only method available but is similar to many other published methods and approaches (7, 8) and commercial protocols (e.g., Micromass, Bruker) as well. Two review articles (7, 8) provide a good overview of this research field as well as a book devoted to this subject of microorganisms analysis by mass spectro-metry that provides additional information and approaches to the analysis of these complex biological samples (9).
Several steps are important for successful MALDI-TOF MS analysis of microorganisms (specifically bacteria). The first step is obtaining relevant microorganism samples for analysis. While only small volumes (|iL) of sample are required for MALDI-TOF MS analysis, currently at least 103 cells are necessary. Analyses of approximately 106 cells per microliter are used more routinely for this direct MALDI-MS analysis. The relative concentrations of matrix and analyte are critical for successful analysis and will be discussed in more detail later. Removal of the growth media from the microorganisms is also important for successful mass spectrometric analysis. A proven and effective way to clean the bacterial samples harvested from liquid growth media is to pellet the cells and wash with water or appropriate volatile solution, such as 2% ammonium chloride, repeatedly. Once adequate bacterial samples are prepared/obtained, the second step is to spot the microbial sample (бее Note 1) onto a MALDI sample plate along with a MALDI matrix compound, which is used to aid in desorption and ionization of intact protein and nonvolatile components within the bacterial cells. There are numerous matrix recipes and spotting procedures in the literature for successful MALDI-MS analysis that are for the most part applicable to analysis of components of microorganisms. The research community currently performing bacterial analysis does not consistently use the same sample matrix and spotting procedures. Provided here is the method we have optimized in our laboratory and have successfully applied to analysis and characterization of vegetative and sporulated bacteria. We have also analyzed fungi with the use of double-stick tape for sampling directly from a fungal colony and applying to the MALDI sample plate (10). After the sample is effectively spotted onto a commercial MALDI sample plate, the sample is analyzed with straightforward instrument parameters. Note that the instrument needs to be well calibrated prior to data collection as with any analysis. The final step is data processing and analysis. PNNL has developed algorithms for justified comparison of sample data with a collected database of MALDI-TOF MS spectra for comparative identification. There are other commercial products available that are designed for use with specific vendor instrumentation and other published approaches to data analysis (9).
Mass spectra can be obtained from unknown bacterial samples and compared with reference spectra to provide information for a correct identification with a computed degree of confidence. Well-defined sample preparation procedures and calibration routines must be adhered to during the collection of replicates for reference spectra. Protein profiles, or fingerprints, obtained from microorganisms by means of MALDI-TOF MS can vary in connection with the choice of solvent system for the matrices (11). Sufficient replication is also necessary to capture minor variations in growth or handling procedures (see Notes 2 and 3). Thereafter, automated statistics-based data analysis algorithms can be used to compile these replicates into a reference spectrum for inclusion into a database (6). This process has also been successfully extended for the identification of microorganisms grown under different growth conditions making it a viable tool in terms of attribution in forensics (12).
Matrix-assisted laser desorption ionization as its name suggests depends on the cocrystallization of the analyte material and matrix molecules (13, 14). A variety of matrices are compatible for use in instruments equipped with a nitrogen laser (337 nm). The ionization process is well suited for mass spectrometric analysis of large biomolecules. The analyte substance is imbedded in the crystallized matrix and irradiated by sufficient laser power to assist in ionization of intact molecules but does not result in fragmentation. Thus, MALDI is often referred to as a soft ioniza-tion technique. MALDI matrices have conjugated ring structures and typically differ only in their attached moieties. Structures for three of the most commonly used matrices for vegetative and sporulated bacteria are shown in Fig. 1.
Each matrix has a unique initial velocity when exposed to a pulsed laser beam under vacuum and as such can provide advantages when interrogating different mass ranges of interest. For example, alpha-cyano-4-hydroxy cinnamic acid (ACHC), often referred to as a hot matrix, has a high initial velocity, and is a preferred matrix for smaller molecules. Sinapinic acid (SA) or 3,5-dimethyoxy-4-hydroxy cinnamic acid and 3-methoxy-4-hydroxy cinnamic acid, commonly referred to as ferulic acid (FA), are more suited for use with most intact vegetative bacterial cells as well as bacterial spore preparations. Typical mass ranges for the ions observed by direct MALDI-MS analysis of the bacterial cells with the protocols described here are 2-20 kDa. A schematic of the preparation of the MALDI sample spot and a scanning electron microscopy image of a portion of the sample spot is provided in Fig. 2.
Once the matrix has been selected, an appropriate solvent system must also be determined. Matrices will be dissolved, usually to the point of saturation, in some ratio of H2 O and organic material. The solvent system is a crucial component of sample preparation and has a major impact both on crystal structure and the degree to which the analytes are incorporated into them.
For the analysis of most readily accessible and ionizable components from intact bacterial cells or spores it is desirable to have the solvent system at a low pH, which is accomplished
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