Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation

Abstract

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice.

Keywords: ambient ionization mass spectrometry; bacterial specie; lipid profile; multivariate statistical analysis; thermal desorption–electrospray ionization/mass spectrometry.

Figure 1

Analytical processes for the direct characterization of microorganisms: (a) a metallic inoculating probe was used to collect a single colony of bacterial species on a Petri dish, (b) analytes on the probe was extracted with organic solvent; (c) analytes on the probe were thermally desorbed and ionized in a TD-ESI source; and (d) mass spectral data of the corresponding bacteria were acquired followed by PCA and HCA. The metallic inoculating probe was then cleaned up by a flame from handheld butane torch. The entire analysis took less than 1 min.

Figure 2

(a) Repeatability test (20 determinations) was performed by TD-ESI/MS to directly analyze single colonies of E. coli, a Gram-negative bacterium. (b) The mass spectrum of blood agar recorded in positive mode. The lipid ions detected from a single colony of E. coli with (c) the 1st (d) the 20th test were detected in positive mode. Product ion mass spectra for (e) m/z 549, (f) m/z 563, and (g) m/z 589 recorded from E coli.

Figure 3

TD-ESI mass spectra recorded in the positive mode of E. faecalis: (a) without any pretreatment, (b) extracted with chloroform/methanol (2:1, v/v) solution, and (c) treated with chloroform/methanol (2:1, v/v) solution containing 5% TFA.

Figure 4

TD-ESI mass spectra recorded in positive mode of Gram-negative bacteria: (a) E. coli, (b) K. pneumonia, (c) M. catarrhalis, (d) P. aeruginosa, and (e) S. marcescens. TD-ESI mass spectra recorded in positive mode of Gram-positive bacteria: (f) B. subtilis, (g) C. striatum, (h) E. faecalis, (i) L. monocytogenes, and (j) S. aureus.

Figure 5

The PCA scores plots (a–c) and HCA (d) of the lipid ion signals on the TD-ESI mass spectra of extracts from 10 bacterial species. Each colony was determined by 10 measurements.

Figure 6

MALDI mass spectra recorded in positive mode for Gram-negative bacteria: (a) E. coli, (b) K. pneumonia, (c) M. catarrhalis, (d) P. aeruginosa, and (e) S. marcescens. MALDI mass spectra recorded in positive mode for Gram-positive bacteria: (f) B. subtilis, (g) C. striatum, (h) E. faecalis, (i) L. monocytogenes, and (j) S. aureus.

Figure 7

The PCA score plot (a), PCA loading plot (b), and HCA (c) of the protein ion signals on the MALDI-TOF mass spectra of extracts from 10 bacterial species. Each colony was determined by 10 measurements.

Figure 8

Time-dependent analysis at a single location within an E. coli colony indicating (a) the correlation between ion signal intensity and incubation time (inset: photos of E. coli colony growing on a Petri dish with time; green dot: sampling spot), and (b) the changes in the ion signal intensity of specific molecules over different durations using TD-ESI/MS. Note: The Y-axis of the line graph was used as an averaged peak area recorded on triplicate experiments to reduce operative variations.