Direct detection of fatty acid ethyl esters using low temperature plasma (LTP) ambient ionization mass spectrometry for rapid bacterial differentiation J. Isabella Zhang, a Anthony B. Costa, a W. Andy Tao ab and R. Graham Cooks * a Received 24th November 2010, Accepted 10th June 2011 DOI: 10.1039/c0an00940g Low temperature plasma mass spectrometry (LTP-MS) was employed to detect fatty acid ethyl esters (FAEE) from bacterial samples directly. Positive ion mode FAEE mass spectrometric profiles of sixteen different bacterial samples were obtained without extraction or other sample preparation. In the range m/z 200–300, LTP mass spectra show highly reproducible and characteristic patterns. To identify the FAEE’s associated with the characteristic peaks, accurate masses were recorded in the full scan mode using an LTQ/Orbitrap instrument, and tandem mass spectrometry was performed. Data were examined by principal component analysis (PCA) to determine the degree of differentiation possible amongst different bacterial species. Gram-positive and gram-negative bacteria are readily distinguished, and 11 out of 13 Salmonella strains show distinctive patterns. Growth media effects are observed but do not interfere with species recognition based on the PCA results. Introduction The application of mass spectrometry (MS) in microbiology for the identification and differentiation of microorganisms is widely established due to its characteristic sensitivity and molecular specificity. Gas chromatography mass spectrometry (GC-MS) 1,2 and pyrolysis mass spectrometry (Py-MS) 3–5 were used to examine bacteria for characteristic fatty acid profiles 6 or small molecules such as sugars. 7 These techniques allow the distinction of gram-positive from gram-negative bacteria as well as more specific species identification based on a comparison of mass spectra with reference spectra. This process is relatively time consuming and requires chemical derivatization steps. More recent applications of mass spectrometry to bacterial identifica- tion have involved the use of matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). Several proteomics- based approaches 8,9 have since been developed for highly accu- rate analysis of peptides and determination of peptide sequences to identify and characterize biomarker proteins for bacterial identification. These reliable approaches involve sample separa- tion procedures, protease digestion, peptide ion fragmentation and database searches, which are labor intensive. MALDI-MS and ESI-MS have also been used to record protein profiles of whole bacteria 10–17 and to obtain abundant, accurate mass ion signals of intact phospholipids in bacteria cells 18,19,20 for bacteria taxonomy. Given the increasing importance of prompt identification of bacteria for food, biosafety and medical analysis, 21,22 ambient ionization methods such as desorption electrospray ionization (DESI) 23–25 and direct analysis in real time (DART) 26 are of substantial interest. These methods provide direct and rapid in situ high throughput analysis. In previous DESI studies of bacteria, 23,25,27,28 a number of fatty acids and phospholipids from the cell lipid bilayer were detected directly from freshly har- vested, untreated bacterial samples, and data were compared by using statistical tools to allow bacterial differentiation. DART has been used to generate fatty acid methyl ester ions from whole bacterial cells, 26 and this may lead to a new approach for microbial identification. A number of ambient ionization methods are based on exposure of the sample to low power plasmas. These include plasma-assisted desorption ionization (PADI), 29 dielectric barrier discharge ionization (DBDI), 30 and flowing atmospheric-pressure afterglow (FAPA), 31,32 as well as low temperature plasma (LTP), 33 which can be used to directly analyze compounds in complex biological samples. 33 The fact that there is no need for any spray solvent 33,34 is the dis- tinguishing feature of the plasma-based methods. The potential of plasma ionization methods for generating ions by which bacterial samples might be characterized has not been reported yet. In previous work, it has been observed that LTP sensitivity is adequate for volatile or semi-volatile molecules, which means the method has the potential to detect microbial fatty acid esters, which may also be useful in bacteria taxonomy. The gold standard method in bacterial taxonomy and classi- fication, fatty acid profiling, is based on fatty acid methyl ester (FAME) determination. 7,35 Routine FAME analysis involves lengthy sample preparation, starting with hydrolysis of bacteria cells, and followed by fatty acid methylation. 13 Gas a Department of Chemistry, Purdue University, West Lafayette, Indiana, 47907, USA. E-mail: cooks@purdue.edu.; Fax: +765-494-9421; Tel: +765-494-5262 b Department of Biochemistry, Purdue University, West Lafayette, Indiana, 47907, USA This journal is ª The Royal Society of Chemistry 2011 Analyst, 2011, 136, 3091–3097 | 3091 Dynamic Article Links C < Analyst Cite this: Analyst, 2011, 136, 3091 www.rsc.org/analyst PAPER Published on 27 June 2011. Downloaded by Dartmouth College Library on 01/04/2017 13:16:17. View Article Online / Journal Homepage / Table of Contents for this issue