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Strain-Level Differentiation of Bacteria by Paper Spray Ionization Mass Spectrometry Casey A Chamberlain, Vanessa Y Rubio, and Timothy J. Garrett Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00330 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019
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Analytical Chemistry
Strain-Level Differentiation of Bacteria by Paper Spray Ionization Mass Spectrometry Casey A. Chamberlain†, Vanessa Y. Rubio‡, Timothy J. Garrett*† †Department
of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida 32610, United States ‡Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States Mass Spectrometry, Paper Spray Ionization, Metabolomics, Bacteria, Oxalobacter formigenes ABSTRACT: Paper spray ionization-mass spectrometry (PSI-MS) is a relatively new analytical technique allowing for rapid mass spectrometric analysis of biological samples with little or no sample preparation. The expeditious nature of the analysis and minimal requirement for sample preparation makes PSI-MS a promising avenue for future clinical assays with one potential application in the identification of different types of bacteria. Although past PSI-MS studies have demonstrated the ability to distinguish between bacteria of different species and morphological classes, achieving within-species strain-level differentiation has never been performed. In this report, we demonstrate the first strain-level bacterial differentiation by PSI-MS with the mammalian intestinal bacterium Oxalobacter formigenes (Oxf). This novel application holds promising clinical significance as it could be used to differentiate between pathogenic bacteria and their harmless commensal relatives, saving time and money in clinical diagnostics. Both whole cells and cell lysates of Oxalobacter strains HC1 and OxWR were analyzed using the Prosolia VeloxTM 360 PSI source coupled to a Thermo Scientific Q Exactive high-resolution mass spectrometer with a rapid 30-sec analytical method. Multivariate statistical analysis followed by examination of significant features provided for and confirmed differentiation between HC1 and OxWR. We report a panel of strain-exclusive metabolites that could serve as potential strain-indicating biomarkers.
Paper spray ionization-mass spectrometry (PSI-MS) is an ambient analytical technique that involves the direct electrospray analysis and measurement of proteins, metabolites, or lipids in biological samples on a porous substrate, often cellulose paper1. A solvent is applied to the substrate with subsequent electrospray ionization for rapid, direct mass spectrometric analysis. The main benefits of PSIMS are the small sample volume, minimal requirements for sample preparation and expedited analysis time2. Typical methods range from 30 sec to 2 min, far less than the runtimes of liquid chromatography-mass spectrometry (LC-MS) based methods, which can extend beyond 20 min per injection for some applications3. The efficiency and user-friendly nature of PSI-MS has generated significant interest in its clinical application potential4, one example being bacterial identification5. In the clinical setting, the diagnosis of bacterial infection is often initially made inexactly by phenotypic examination of the patient or general tests for inflammation or high white blood cell count6, leading to the prescription of general antibiotics that damage the diversity of the microbiome7. When an exact determination of the causative agent of infection is necessary, further investigation is conducted to identify the pathogen. Among other methods, two of the most common approaches are standard culture techniques and serological antigen tests. Clinical cultures can take as long as 48-72 hours and can easily become contaminated with poor sampling techniques6. Additionally, there are many pathogens which cannot be cultured using routine methods6,8. Serological antigen tests are usually rapid, but lack in sensitivity and cannot always conclusively rule out
infection6. They also often have low specificity due to offtarget reactivity with other antigens and can generate falsenegative results if the infection is in an early stage6,9. These issues spanning from cost to assay confidence to time requirements indicate the need for a rapid, reliable assay for the differentiation and identification of bacteria. To address this need, various MS-based applications have been investigated in regard to bacterial identification as early as 197510-13. Currently, matrix-assisted laser desorption ionization (MALDI) is used as a clinical MS method for bacterial analysis on the basis of protein biomarker detection 14. MALDI-MS shares many benefits with PSI-MS in being rapid, economical, and sensitive, although one advantage of PSI-MS is perhaps the absence of sample preparation compared to MALDI-MS, which requires proper application of a matrix to the biological sample for reproducible analysis2,15. Only in the last decade has PSI-MS been applied to bacterial identification. Previous PSI-MS applications have demonstrated the ability to discern between bacteria of different species and morphological classes (Gram-positive versus Gram-negative)5. However, there are many species of bacteria where only one strain or a small percentage of strains within that species are pathogenic16. A well-known example is Escherichia coli (E. coli), a species with many strains that exist naturally in the intestine, but which contains a subset of pathogenic strains such as the notorious O157:H7, which causes over 73,000 illnesses per year in the United States alone17,18. Identification of bacteria at the strain level often requires expensive and time-consuming genomic methods, and there is a great need for improved techniques in this area of
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diagnostic medicine19,20. A PSI-MS method with the capability to distinguish between common pathogens and their samespecies nonpathogenic relatives could revolutionize the diagnosis of infectious disease by providing a rapid, routine pipeline to determine the cause of infection. The cornerstone of such an assay would be the detection of strain-exclusive biomarkers for confident identification of a specific strain within a specific species of bacteria. To date, no PSI-MS method has demonstrated the capability to differentiate between different strains of the same species of bacteria. This report describes the first PSI-MS application achieving strainlevel differentiation of bacteria. Using the commensal mammalian intestinal bacterium Oxalobacter formigenes as the experimental model, an oxalate-degrading microorganism currently being investigated by our laboratory as a future probiotic therapy for kidney stones and other oxalate diseases21, we demonstrate this advancement in PSI-MS application showing analytical distinction between two of its strains, HC1 and OxWR, to better characterize their biological differences. EXPERIMENTAL SECTION Cell Culture and Sample Generation Pure cultures of Oxf HC1 and OxWR were grown anaerobically from frozen glycerol stocks. For each strain, 0.5 mL stock was used to inoculate 75 mL of previously published Oxf-specific media supplemented with 100 mM oxalate22. Cultures were incubated at 37C for 72 hours, after which 875 mL bottles of Oxf media were inoculated with 5 mL culture, yielding 8 culture replicates per strain. Cultures were incubated for 48 hours, after which cell pellets for each strain were combined by sequential centrifugation of replicate cultures at 15,000 × g, 4°C for 5 min, discarding the supernatant after each addition. Pellets were washed 3 times by resuspension in 6mL 20 mM ammonium acetate with centrifugation and discarding of supernatant between all washes. In the third wash cycle, cell resuspensions were transferred to pre-weighed 20 mL vials, centrifuged, dried, and weighed for downstream normalization. Pellets were resuspended in 6 mL 20 mM ammonium acetate and 1 mL resuspension was removed and frozen at -80°C for whole cell analysis. Remaining resuspension was transferred to 15 mL polypropylene vials chilled in an ice slurry water bath for sonication. Cells were lysed using a Sonic Dismembrator Model 500 with a Branson Sonicator Probe (Thermo Fisher Scientific, Waltham, MA, USA) by the following method: 30% amplitude for 30 sec, 1 min cool-down, 60% amplitude for 30 sec, 2 min cool-down, 60% amplitude for 15 sec. Lysates were immediately frozen at -80°C. PSI-MS Instrumentation and Analysis Lysate and whole cell samples for Oxf HC1 and OxWR were thawed on ice and normalized based on cell concentration, adjusting volume with 20mM ammonium acetate. For both analyses, 15 µL sample was pipetted onto VeloxTM sample cartridges (Prosolia Inc., Indianapolis, IN) containing pre-cut triangular analysis paper using a custom 3D-printed stabilizing device (Prosolia Inc., Indianapolis, IN) for reproducibility in sample dispensing and positioning. Samples were loaded into the Prosolia VeloxTM 360 (Prosolia Inc., Indianapolis, IN) connected to a Thermo Scientific Q Exactive Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA) for analysis in alternating sequence order by strain. 4:1 H2O:Acetonitrile containing 0.1% formic acid was used as the wetting and spray
solvent with 80 µL applied for wetting. This solvent was chosen to match the mobile phase gradient composition from past related LC-MS experiments with sufficient elution of both hydrophobic and hydrophilic species21. Spray voltage for ionization was 4.5 kV. Data acquisition was performed in positive ion mode at 140,000 mass resolution for 30 sec after 9 sec equilibration. Scan range was 70-1000 m/z. Additional instrumentation and analysis parameters are provided in Table S1. Acquired data were quality-checked for analytical reproducibility using the total ion current (TIC) both across the method duration and from sample-to-sample. Raw data TIC relative standard deviation (RSD) below 10% was observed across all samples in the HC1 lysates (9.1%), OxWR lysates (9.9%), and HC1 whole cell samples (5.2%). OxWR whole cell samples showed notable variance in raw data TIC (RSD=52.3%) without clear explanation. However, this variance was reduced to
