MSACL 2016 US Abstract

Metal Oxide Laser Ionization (MOLI) MS for Identification of Bacteria Using Fatty Acid Profiling

Kent Voorhees (Presenter)
Colorado School of Mines

Bio: Dr. Kent Voorhees is Research Professor of Chemistry at the Colorado School of Mines (CSM). He has been a faculty member at CSM for 36 years and has received many awards including the Dean's Excellence Award and the Board of Trustees Outstanding Faculty Award. His research has been in the areas of environmental and microbiological mass spectrometry. His research group was a key player in the development of the US Army's Chemical and Biological Mass Spectrometer Block I and II. Kent and Ricky Holland conducted the first experiments on using MALDI MS for identification of whole cell bacteria using protein profiles. Dr. Voorhees is now promoting bacterial identification using metal oxide mass spectrometry (MOLI MS) to generate fatty acid profiles. He holds 18 patents and has published over 150 publications. Dr. Voorhees is the editor of the Journal of Analytical Pyrolysis.

Authorship: Kent J. Voorhees, Nickolas R. Saichek, Kirk R. Jensen, Casey McAlpin and Christopher R. Cox
Colorado School of Mines

Short Abstract

Recently, matrix-assisted laser desorption ionization MS (MALDI MS) instruments were utilized with metal oxides as a matrix replacement and catalyst to cleave bacterial lipid extracts to their constituent fatty acids. Utilizing this novel approach designated metal oxide laser ionization (MOLI) MS, with CaO, NiO, and CeO2, fatty acids and lipid material from bacterial membrane lipids in the source of the MS were produced, resulting in profiles that were used to identify microorganisms. The data from diverse bacterial phenotypes have shown that MOLI MS is a strain-level detection system providing leave-one-out cross validation results greater than 98 percent. Organism classification results from several metal oxides will be presented.

Long Abstract

Mass spectrometry has been become ubiquitous and is constantly updating itself. In this presentation, we describe an extension of matrix-assisted laser desorption ionization MALDI MS for the analysis of small molecule (MW< 1000 Da) lipid compounds using metal oxide nano-particles as a matrix free platform. The resulting spectra are free of background peaks and provide an M+1 or M-1 peak for common lipid materials. Several metal oxide compounds were studied; thus far NiO<100> demonstrated the highest peak counts and signal to noise ratio for molecular ions. Other metal oxides were found to catalytically cleave lipid materials into their constituent fatty acids. BaO, SrO, Na2O, CaO, Rb2O, MgO, CeO2, and Cs2O have been investigated for lipid cleavage, stability, activation, and activity to both chloroform extracts and whole bacterial cells. Metal oxide MALDI MS has been designated as MOLI MS.

Utilizing MOLI MS in both positive- and negative-ion modes with CaO, fatty acids from bacterial membrane lipids in the source of the MS were produced, resulting in profiles that could be used to identify the microorganism. The data from 10 unrelated bacterial phenotypes, Bacillus anthracis, Clostridium putrefaciens, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Francisella tularensis, Salmonella typhimurium, Yersinia pestis, showed that replicates of each organism grouped in a unique principal component space. Data were collected on a Bruker UltrafleXtreme mass spectrometer equipped with the smartbeam-II laser(Nd:YAG at 355nm, 100AJ/shot) with 1kHz repetition rate. Fatty acid profile data were analyzed by principal components analysis and leave-one-out cross validation using the R statistics package. Cross validation results of 94-97 % % were obtained for the 10 bacterial species.

In evaluating the stability of CaO, it was found that the bacterial profiles changed as a function of time from the initial thermal activation and was clearly unacceptable for use in a clinical laboratory. CeO2 in the negative-ion mode was eventually shown to best fit the above catalyst criteria. Fatty acid profiles from four sample sets of the 10 previously used bacteria were collected with stored catalyst at 0, 8, 24, and 504 h. ANOVA showed no effect based on long-term catalyst degradation. Principal component analysis provided confirmation that the four sample sets were independent of the post-activation age of the catalyst. In this plot, each individual category regardless of storage time, plotted in the same space. The four10 bacteria data sets provided leave-one-out cross validation results greater than 98 percent.

Bacteria analyzed with MOLI MS using CeO2 in the negative-ion mode produce more reproducible and higher ion intensity fatty acid profiles. Positive- ion data can be obtained, but requires a highly trained operator. The positive ion data did not show the ion intensity observed in negative-ion mode.

A standard operating protocol (SOP) considering the effect of growing conditions, storage time, and extraction procedures will be discussed. Results from application of MOLI MS to classify the 10 bacteria discussed above along with support material for the SOP will be presented. For bacterial classifications, MOLI MS using CeO2 has proven to be a strain-level detection system. This will be demonstrated using a study of 54 Staphylococcus isolates. Cross validation (CV) indicated accuracies of 98% and 96% at the species and strain levels, respectively. These results were comparable to protein-profiling methods at the species level while offering strain-level identification.

References & Acknowledgements:

Background References:

Casey R. McAlpin, Kent J. Voorhees, April R. Corpuz and Ryan Richards, (2012) “Analysis of Lipids: Metal Oxide Laser Ionization Mass Spectrometry.” Anal. Chem., 84, 7677

K.J. Voorhees, K.R. Jensen, C.R. McAlpin, C.R. Cox, J.C. Rees, R. Cody and M. Ubukata (2013) “Modified MALDI MS Lipid Profiling for Identification of Bacteria,” J. Mass Spectrometry, 48 850.

K.J. Voorhees, N.R. Saichek, K.R. Jensen, P.B. Harrington and C.R. Cox, (2014) “Catalytic Pyrolysis for Bacterial Identification.” J. Anal. Appl. Pyrol. DOI: 10.1016/j.jaap.2014.10.016.

C.R. Cox, N.R. Saichek, K.R. Jensen, P.B. Harrington and K.J. Voorhees, (2015) “Strain-level bacterial identification by CeO2-catalyzed MALDI-TOF MS fatty acid analysis and comparison to commercial protein-based methods”, Nature: Scientific Reports Scientific Reports, 5, Article number:10470 doi:10.1038/srep10470.

Acknowledgement- The authors wish to thank the National Science Foundation (CHE-1229156) for partial support of this study.

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