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RESEARCH PROFILES Accurately identifying bacteria Identifying bacteria by microscopy is a slow process. Bacteria must be cultured, and then significant human expertise and experience are needed to categorize them. A succession of MS techniques have been used to streamline the process; the latest, developed by Charles Wilkins and colleagues at the University of Arkansas and presented in the March 15 issue of Analytical Chemistry (pp 1340–1347), combines MALDI and FTMS to analyze the contents of whole bacteria. Wilkins’s group accurately determined masses for E. coli proteins with errors of >27 ppm for several ribosomal proteins that could be used as taxonomic fingerprints for the bacteria. Such information could be used to speed medical diagnoses or assess biological warfare threats. MALDI has been used by other groups to examine whole bacteria because the technique reduces the sample preparation time by eliminating the need for prior separations of cell contents. Bacteria are then identified by comparing the measured protein masses with calculated masses. In addition, fewer cells need to be cultured if they are being examined by microscope. However, these whole-cell MALDI techniques typically used TOFMS, which lacks the resolution and precision to identify proteins with very similar masses. Therefore, Wilkins and colleagues turned to the much more accurate FTMS. “Obviously, the less certainty you have about the mass, the more possibilities arise,” says Wilkins. “The better you know the mass, the easier it is to eliminate other possibilities.” As with many scientific endeavors, Wilkins began his investigation not with the goal to improve the bacterial identification process, but to examine “coldshock proteins”, which are thought to form when microbes are subjected to temperatures around 10 °C as they grow. “In these previous studies, various people have reported that they identified different cold-shock proteins, but they were all within the experimental error
P02436 m/z 6254
100 90 80 70 60 50
P02435 m/z 6315
P28690 m/z 5095
P02430 m/z 6410
40
P02378 m/z 9535 P02372 m/z 9190
30
P02429 m/z 7274
20 10 0
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FTMS spectrum of E. coli whole cell with proteins identified on the basis of ion mass.
of the same mass,” says Wilkins. Using FTMS instead of TOF, Wilkins’s group called into question the presence of some cold-shock proteins. “Originally, we expected that we would see some of these cold-shock proteins,” says Wilkins. “But as we looked into it further and made more accurate measurements, we discovered we were seeing ribosomal proteins.” For instance, the ribosomal protein P02429 (SWISSPROT reference number) is very close in mass to cold-shock proteins CspA (7267.58808 Da) and CspC (7266.7211 Da). Using FTMS with cold-shocked and normal E. coli, researchers consistently saw a peak at 7274.196 Da, which differs in mass from the SWISSPROT value for P02429 by only 0.195 Da. A less-accurate TOF instrument would not be able to distinguish between these three possible proteins. In addition, Wilkins’s group determined that P02429 retains its terminal methionine residue, which is unusual in bacteria. The researchers also matched seven other ion peaks, between 5095 and 9535 Da, with proteins in the database.
They found another protein, P28690, which retained methionine and one protein, P02436, which was methylated. Similar results were found, but not proven, by TOFMS, they say. While cold-shock proteins may exist under specific conditions, the ribosomal proteins, which account for more than 20% of an organism’s proteins, are produced in all stages of a bacteria’s growth. “We would like to have a set of proteins that are more or less reproducible,” says Wilkins. “Because ribosomal proteins are in such abundance, it’s more likely you will find them than some other unusual proteins that may only occur at one stage of the growth.” In addition, ribosomal proteins occur in significant quantities on cell surfaces and in the cell wall, making them more likely to be ablated and ionized by the laser in MALDI. Wilkins believes that FTMS, “should become the method of choice for MALDI-MS analysis of intact microorganisms in studies for which unambiguous identification of targeted proteins is the expected outcome.” —Michael Felton
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