ESI FTICR mass spectrometry is the only technique currently used for accurate molecular weight analysis of PCR products above 100 bp in size. This is important in demonstrating the potential for MS in making major contributions in the molecular biology and genomics areas. In the near future, it is more likely that less expensive, more user friendly MS techniques will be used for high-throughput analyses (including MALDI TOF and ESI quadrupole). There have been numerous reports on the use of MALDI TOF. The current report is to the first to evaluate the use of ESI-quadrupole analysis of PCR products. Synthetic oligonucleotides (30 and 89 mers) and polymerase chain reaction products of varying molecular weight (62, 88, 89, and 114 bp) were analyzed by ESI using a quadrupole MS. The mass accuracy for nucleic acids in the 30-62 bp range was shown to allow determination of nucleotide substitutions and additions/deletions. For higher molecular weight PCR products (88-114 bp), the mass accuracy of ESI-MS distinguishes single or multiple nucleotide insertions/deletions. In addition, ESI quadrupole MS allows determination of molecular weight of both strands of higher molecular weight ds PCR products and can distinguish nucleotide modifications (e.g., with biotin). In conclusion, it is demonstrated that ESI-MS occupies an intermediate position (as compared to MALDI TOF and ESI FTICR) with regard to mass accuracy and resolution in analysis of nucleic acids.

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

Bacterial cell envelope components are widely distributed in airborne dust, where they act as inflammatory agents causing respiratory symptoms. Measurements of these agents and other environmental factors are assessed in two elementary schools in a southeastern city in the United States. Muramic acid (MA) was used as a marker for bacterial peptidoglycan (PG), and 3-hydroxy fatty acids (3-OH FAs) were used as markers for Gram-negative bacterial lipopolysaccharide (LPS). Culturable bacteria were collected using an Andersen sampler with three different culture IMPLICATIONS Levels of bacteria and their constituents (PG and LPS) are correlated with total surface dust and TSP levels in school environments. This suggests an important environmental health guideline for school administrators: decreasing both surface dust and TSP levels will likely reduce potential exposure to harmful biocontaminants. The high correlations between CO 2 and levels of bacteria and their constituents also indicate that an appropriate air-exchange rate may help minimize biocontamination. The analytical MA and 3-OH FA methods measure culturable and nonculturable bacteria as well as bacterial cell wall remnants, whereas the culture method only assesses the culturable portion of the bacterial population. Together, these three different measures provide complementary information for characterizing biocontamination of indoor air, which is more informative than information provided by culture alone. Additional evaluation of other environmental factors, including CO 2 and air-exchange rate, might suggest recommendations for lowering biocontamination in buildings.media. In addition, temperature (T), relative humidity (RH), and CO 2 were continuously monitored.Concentrations of airborne MA and 3-OH FAs were correlated with total suspended particulate (TSP) levels. Outdoor MA (mean = 0.78-1.15 ng/m 3 ) and 3-OH FA levels (mean = 2.19-2.18 ng/m 3 ) were similar at the two schools. Indoor concentrations of airborne MA and 3-OH FAs differed significantly between schools (MA: 1.44 vs. 2.84 ng/m 3 ; 3-OH FAs: 2.96 vs. 4.57 ng/m 3 ). Although indoor MA levels were low, they were significantly related to teachers' perception of the severity of indoor air quality (IAQ) problems in their classrooms. Concentrations of CO 2 correlated significantly with all bacteria measurements. Because CO 2 levels were related to the number of occupants and the ventilation rates, these findings are consistent with the hypothesis that the children and teachers are sources of bacterial contamination. Many culturable bacteria present in indoor air are opportunistic organisms that can be infectious for compromised individuals, while both culturable and nonculturable bacterial remnants act as environmental toxins for both healthy and compromised individuals. Measuring the "total bacteria load" would be most accurate in assessing the biotoxicity of indoor air. Chemical analysis of MA and 3-OH FAs, when coupled with the conventional culture method, provides complementary infor...

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Muramic acid serves as a marker for the presence of bacterial cell wall debris in mammalian tissues. There have been a number of controversial and sometimes conflicting results on assessing the levels of muramic acid in health and disease. The present report is the first to use the state-of-the art technique, gas chromatographytandem mass spectrometry, to identify and quantify the levels of muramic acid in tissues. Muramic acid was not found in normal rat brain or spleen. However, when tissues were spiked with muramic acid, it was readily identified. The detection limit was <1 ng of muramic acid/100 mg (wet weight) of tissue. The levels of muramic acid reported in diseased human spleen and spleen of arthritic rats, previously injected with bacterial cell walls, were 100-to 1,000-fold higher. In the present study, muramic acid was also readily detected in the cerebrospinal fluid of patients with pneumococcal meningitis (6.8 to 3,900 ng of muramic acid/ml of cerebrospinal fluid). In summary, there can be an enormous difference in the levels of muramic acid found in different mammalian tissues and body fluids in health and disease. This report could have great impact in future studies assessing the role of bacterial cell wall remnants in the pathogenesis of certain human inflammatory diseases.

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