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Bacterial identification by lipid profiling using liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

  • Sophie E. Lellman and Rainer Cramer EMAIL logo
Published/Copyright: December 19, 2019
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 58 Issue 6

Abstract

Background

In recent years, mass spectrometry (MS) has been applied to clinical microbial biotyping, exploiting the speed of matrix-assisted laser desorption/ionization (MALDI) in recording microbe-specific MS profiles. More recently, liquid atmospheric pressure (AP) MALDI has been shown to produce extremely stable ion flux from homogenous samples and ‘electrospray ionization (ESI)-like’ multiply charged ions for larger biomolecules, whilst maintaining the benefits of traditional MALDI including high tolerance to contaminants, low analyte consumption and rapid analysis. These and other advantages of liquid AP-MALDI MS have been explored in this study to investigate its potential in microbial biotyping.

Methods

Genetically diverse bacterial strains were analyzed using liquid AP-MALDI MS, including clinically relevant species such as Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Bacterial cultures were subjected to a simple and fast extraction protocol using ethanol and formic acid. Extracts were spotted with a liquid support matrix (LSM) and analyzed using a Synapt G2-Si mass spectrometer with an in-house built AP-MALDI source.

Results

Each species produces a unique lipid profile in the m/z range of 400–1100, allowing species discrimination. Traditional (solid) MALDI MS produced spectra containing a high abundance of matrix-related clusters and an absence of lipid peaks. The MS profiles of the bacterial species tested form distinct clusters using principle component analysis (PCA) with a classification accuracy of 98.63% using a PCA-based prediction model.

Conclusions

Liquid AP-MALDI MS profiles can be sufficient to distinguish clinically relevant bacterial pathogens and other bacteria, based on their unique lipid profiles. The analysis of the lipid MS profiles is typically excluded from commercial instruments approved for clinical diagnostics.

Keywords: bacteria; biotyping; lipids; mass spectrometry; matrix-assisted laser desorption/ionization (MALDI); profiling; speciation
Corresponding author: Prof. Rainer Cramer, Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, UK, Phone: +44-118-378-4550

Funding source: EPSRC

Award Identifier / Grant number: EP/R513301/1

Funding statement: This work was supported by EPSRC through grant EP/R513301/1, Funder Id: http://dx.doi.org/10.13039/501100000266.

Acknowledgments

Access to the AMX [Beta] software was kindly provided by Waters Corporation.

  1. Author contributions: All authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Employment or leadership: None declared.

  3. Honorarium: None declared.

  4. Competing interests: The funding organization played no role in the study design; in the collection, analysis, and interpretation of data; in writing of the report; or in the decision to submit the report for publication.

References

1. Carbonnelle E, Mesquita C, Bille E, Day N, Dauphin B, BerettiJ-L, et al. MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin Biochem 2011;44:104–9.10.1016/j.clinbiochem.2010.06.017Search in Google Scholar PubMed

2. Croxatto A, Prod’hom G, Greub G. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS Microbiol Rev 2012;36:380–407.10.1111/j.1574-6976.2011.00298.xSearch in Google Scholar PubMed

3. Perez KK, Olsen RJ, Musick WL, Cernoch PL, Davis JR, Land GA, et al. Integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs. Arch Pathol Lab Med 2013;137:1247–54.10.5858/arpa.2012-0651-OASearch in Google Scholar PubMed

4. Patel TS, Kaakeh R, Nagel JL, Newton DW, Stevenson JG. Cost analysis of implementing matrix-assisted laser desorption ionization–time of flight mass spectrometry plus real-time antimicrobial stewardship intervention for bloodstream infections. J Clin Microbiol 2016;55:60–7.10.1128/JCM.01452-16Search in Google Scholar PubMed PubMed Central

5. Faron ML, Buchan BW, Hyke J, Madisen N, Lillie JL, Granato PA, et al. Multicenter evaluation of the Bruker MALDI biotyper CA system for the identification of clinical aerobic gram-negative bacterial isolates. PLoS One 2015;10:e0141350.10.1371/journal.pone.0141350Search in Google Scholar PubMed PubMed Central

6. Seng P, Drancourt M, Gouriet F, Scola BL, Fournier PE, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 2009;49:543–51.10.1086/600885Search in Google Scholar PubMed

7. Han X, Gross RW. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 2003;44:1071–9.10.1194/jlr.R300004-JLR200Search in Google Scholar PubMed

8. Dworzanski JP, Berwald L, Meuzelaar HL. Pyrolytic methylation-gas chromatography of whole bacterial cells for rapid profiling of cellular Fatty acids. Appl Environ Microbiol 1990;56:1717–24.10.1128/aem.56.6.1717-1724.1990Search in Google Scholar PubMed PubMed Central

9. Basile F, Voorhees KJ, Hadfield TL. Microorganism gram-type differentiation based on pyrolysis-mass spectrometry of bacterial Fatty Acid methyl ester extracts. Appl Environ Microbiol 1995;61:1534–9.10.1128/aem.61.4.1534-1539.1995Search in Google Scholar PubMed PubMed Central

10. Dworzanski JP, Tripathi A, Snyder AP, Maswdeh WM, Wick CH. Novel biomarkers for Gram-type differentiation of bacteria by pyrolysis–gas chromatography–mass spectrometry. J Anal Appl Pyrol 2005;73:29–38.10.1016/j.jaap.2004.09.003Search in Google Scholar

11. Fang J, Barcelona MJ. Structural determination and quantitative analysis of bacterial phospholipids using liquid chromatography/electrospray ionization/mass spectrometry. J Microbiol Methods 1998;33:23–35.10.1016/S0167-7012(98)00037-2Search in Google Scholar

12. Smith PB, Snyder AP, Harden CS. Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry. Anal Chem 1995;67:1824–30.10.1021/ac00107a011Search in Google Scholar PubMed

13. Gidden J, Denson J, Liyanage R, Ivey DM, Lay JO. Lipid compositions in Escherichia coli and Bacillus subtilis during growth as determined by MALDI-TOF and TOF/TOF mass spectrometry. Int J Mass Spectrom 2009;283:178–84.10.1016/j.ijms.2009.03.005Search in Google Scholar PubMed PubMed Central

14. Hamid AM, Jarmusch AK, Pirro V, Pincus DH, Clay BG, Gervasi G, et al. Rapid discrimination of bacteria by paper spray mass spectrometry. Anal Chem 2014;86:7500–7.10.1021/ac501254bSearch in Google Scholar PubMed

15. Heller DN, Cotter RJ, Fenselau C, Uy OM. Profiling of bacteria by fast atom bombardment mass spectrometry. Anal Chem 1987;59:2806–9.10.1021/ac00150a018Search in Google Scholar PubMed

16. Strittmatter N, Rebec M, Jones EA, Golf O, Abdolrasouli A, Balog J, et al. Characterization and identification of clinically relevant microorganisms using rapid evaporative ionization mass spectrometry. Anal Chem 2014;86:6555–62.10.1021/ac501075fSearch in Google Scholar PubMed

17. Shu X, Li Y, Liang M, Yang B, Liu C, Wang Y, et al. Rapid lipid profiling of bacteria by online MALDI-TOF mass spectrometry. Int J Mass Spectrom 2012;321–322:71–6.10.1016/j.ijms.2012.05.016Search in Google Scholar

18. Cramer R, Pirkl A, Hillenkamp F, Dreisewerd K. Liquid AP-UV-MALDI enables stable ion yields of multiply charged peptide and protein ions for sensitive analysis by mass spectrometry. Angew Chem Int Ed Engl 2013;52:2364–7.10.1002/anie.201208628Search in Google Scholar PubMed PubMed Central

19. Towers MW, Mckendrick JE, Cramer R. Introduction of 4-Chloro-α-cyanocinnamic acid liquid matrices for high sensitivity UV-MALDI MS. J Proteome Res 2010;9:1931–40.10.1021/pr901089jSearch in Google Scholar PubMed

20. Ryumin P, Brown J, Morris M, Cramer R. Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS. Methods 2016;104:11–20.10.1016/j.ymeth.2016.01.015Search in Google Scholar PubMed

21. Ryumin P, Cramer R. The composition of liquid atmospheric pressure matrix-assisted laser desorption/ionization matrices and its effect on ionization in mass spectrometry. Anal Chim Acta 2018;1013:43–53.10.1016/j.aca.2018.01.070Search in Google Scholar PubMed

22. Trimpin S, Wang B, Inutan ED, Li J, Lietz CB, Harron A, et al. A mechanism for ionization of nonvolatile compounds in mass spectrometry: considerations from MALDI and inlet ionization. J Am Soc Mass Spectrom 2012;23:1644–60.10.1007/s13361-012-0414-ySearch in Google Scholar PubMed

23. Hale OJ, Cramer R. Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions. Anal Bioanal Chem 2018;410:1435–44.10.1007/s00216-017-0788-6Search in Google Scholar PubMed

24. Hale OJ, Morris M, Jones B, Reynolds CK, Cramer R. Liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry adds enhanced functionalities to MALDI MS profiling for disease diagnostics. ACS Omega 2019;4:12759–65.10.1021/acsomega.9b01476Search in Google Scholar PubMed PubMed Central

25. Hale OJ, Ryumin P, Brown JM, Morris M, Cramer R. Production and analysis of multiply charged negative ions by liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2018;1–8. https://doi.org/10.1002/rcm.8246.10.1002/rcm.8246Search in Google Scholar PubMed PubMed Central

26. Bruker Daltonik GmbH. Instructions for Use – MALDI Biotarget 48. https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/InstructionForUse/IFU_268711_267615_226413_MALDI_Biotarget_48_Rev1.pdf.Search in Google Scholar

27. Public Health England. Culture Collections [National Collection of Type Cultures]. https://www.phe-culturecollections.org.uk/collections/nctc.aspx.Search in Google Scholar

28. Cramer R, Corless S. The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics. Rapid Commun Mass Spectrom 2001;15:2058–66.10.1002/rcm.485Search in Google Scholar PubMed

29. Marty MT, Baldwin AJ, Marklund EG, Hochberg GK, Benesch JL, Robinson CV. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal Chem 2015;87:4370–6.10.1021/acs.analchem.5b00140Search in Google Scholar PubMed PubMed Central

30. Smirnov IP, Zhu X, Taylor T, Huang Y, Ross P, Papayanopoulos IA, et al. Suppression of α-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry. Anal Chem 2004;76:2958–65.10.1021/ac035331jSearch in Google Scholar PubMed

31. Vieler A, Wilhelm C, Goss R, Süß R, Schiller J. The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC. Chem Phys Lipids 2007;150:143–55.10.1016/j.chemphyslip.2007.06.224Search in Google Scholar PubMed

32. Jackson SN, Wang H-Y, Woods AS. Direct profiling of lipid distribution in brain tissue using MALDI-TOFMS. Anal Chem 2005;77:4523–7.10.1021/ac050276vSearch in Google Scholar PubMed

33. Cerruti CD, Benabdellah F, Laprévote O, Touboul D, BrunelleA. MALDI imaging and structural analysis of rat brain lipid negative ions with 9-aminoacridine matrix. Anal Chem 2012;84:2164–71.10.1021/ac2025317Search in Google Scholar PubMed

34. Angelini R, Vitale R, Patil VA, Cocco T, Ludwig B, Greenberg ML, et al. Lipidomics of intact mitochondria by MALDI-TOF/MS. J Lipid Res 2012;53:1417–25.10.1194/jlr.D026203Search in Google Scholar PubMed PubMed Central

35. Angelini R, Babudri F, Lobasso S, Corcelli A. MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine. J Lipid Res 2010;51:2818–25.10.1194/jlr.D007328Search in Google Scholar PubMed PubMed Central

36. Structure Database (LMSD) LIPID MAPS (R) Lipidomics Gateway. https://www.lipidmaps.org/data/structure/LMSDSearch.php?Mode=SetupTextOntologySearch.Search in Google Scholar

37. Zhang JI, Talaty N, Costa AB, Xia Y, Tao WA, Bell R, et al. Rapid direct lipid profiling of bacteria using desorption electrospray ionization mass spectrometry. Int J Mass Spectrom 2011;301: 37–44.10.1016/j.ijms.2010.06.014Search in Google Scholar

38. Woods AS, Ugarov M, Egan T, Koomen J, Gillig KJ, Fuhrer K, et al. Lipid/peptide/nucleotide separation with MALDI-ion mobility-TOF MS. Anal Chem 2004;76:2187–95.10.1021/ac035376kSearch in Google Scholar PubMed

39. Venne K, Bonneil E, Eng K, Thibault P. Improvement in peptide detection for proteomics analyses using NanoLC−MS and high-field asymmetry waveform ion mobility mass spectrometry. Anal Chem 2005;77:2176–86.10.1021/ac048410jSearch in Google Scholar PubMed

40. Barák I, Muchová K. The role of lipid domains in bacterial cell processes. Int J Mol Sci 2013;14:4050–65.10.3390/ijms14024050Search in Google Scholar PubMed PubMed Central

41. Pendleton JN, Gorman SP, Gilmore BF. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther 2013;11: 297–308.10.1586/eri.13.12Search in Google Scholar PubMed

42. Ishida Y, Madonna AJ, Rees JC, Meetani MA, Voorhees KJ. Rapid analysis of intact phospholipids from whole bacterial cells by matrix-assisted laser desorption/ionization mass spectrometry combined with on-probe sample pretreatment. Rapid Commun Mass Spectrom 2002;16:1877–82.10.1002/rcm.802Search in Google Scholar PubMed

43. Ishida Y, Kitagawa K, Nakayama A, Ohtani H. Complementary analysis of lipids in whole bacteria cells by thermally assisted hydrolysis and methylation-GC and MALDI-MS combined with on-probe sample pretreatment. J Anal Appl Pyrol 2006;77:116–20.10.1016/j.jaap.2006.02.006Search in Google Scholar

44. Peschel A, Jack RW, Otto M, Collins LV, Staubitz P, Nicholson G, et al. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine. J Exp Med 2001;193:1067–76.10.1084/jem.193.9.1067Search in Google Scholar PubMed PubMed Central

45. Calvano CD, Zambonin CG, Palmisano F. Lipid fingerprinting of Gram-positive lactobacilli by intact cells – matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge based matrix. Rapid Commun Mass Spectrom 2011;25:1757–64.10.1002/rcm.5035Search in Google Scholar PubMed

46. Freiwald A, Sauer S. Phylogenetic classification and identification of bacteria by mass spectrometry. Nat Protoc 2009;4:732.10.1038/nprot.2009.37Search in Google Scholar PubMed

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0908).

Received: 2019-08-27
Accepted: 2019-11-25
Published Online: 2019-12-19
Published in Print: 2020-06-25

©2019 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2019-0908
Keywords for this article
bacteria; biotyping; lipids; mass spectrometry; matrix-assisted laser desorption/ionization (MALDI); profiling; speciation

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Advancements in mass spectrometry as a tool for clinical analysis: part II
  4. Quantitative protein assessment
  5. Complexity, cost, and content – three important factors for translation of clinical protein mass spectrometry tests, and the case for apolipoprotein C-III proteoform testing
  6. Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation
  7. Development and evaluation of an element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection: can we apply the new assay in the clinical laboratory?
  8. MALDI-MS for the clinic
  9. Matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS): basics and clinical applications
  10. Clinical use of mass spectrometry (imaging) for hard tissue analysis in abnormal fracture healing
  11. Cellular resolution in clinical MALDI mass spectrometry imaging: the latest advancements and current challenges
  12. Bacterial identification by lipid profiling using liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry
  13. Clinical application of ’omics technologies
  14. Individualized metabolomics: opportunities and challenges
  15. Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre
  16. The “olfactory fingerprint”: can diagnostics be improved by combining canine and digital noses?
  17. Peptidomic and proteomic analysis of stool for diagnosing IBD and deciphering disease pathogenesis
  18. The influence of hypoxia on the prostate cancer proteome
  19. Laboratory automation and kit-based approaches
  20. Mass spectrometry and total laboratory automation: opportunities and drawbacks
  21. The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?
  22. Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory
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