Page 8 - Microorganism Species Analysis
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LCMS-2020                                                                                                                                                                                                           LCMS-2020
                                                            Low-Molecular Information
            Microorganism Solutions                                                                                                                                                                                              Microorganism Solutions




                     Microorganism Classification by Chemical Analysis                                                                    Data
                     In addition to gene analysis or cultivation, staining, and shape observation, microorganisms can also be classified by the
                     chemical analysis of the microorganism components. Methods previously known for the classification of microorganisms   Analysis of the Actinomycete Menaquinone Using LC/MS
                     include using the guanine-cytosine content of the constituent DNA as an index for the microorganism genome and the GC or
                                                                                                                                           The analysis of the bacterial fatty acid composition and isoprenoid quinones has conventionally been used as a   Microorganism Species  Observation of
                     GC/MS analysis of the fatty acid cell membrane components after extraction and derivatization.
                                                                                                                                           chemical classification indicator for actinomycetes.
                     LC/MS is a powerful tool for the analysis and taxonomic evaluation of compounds such as isoprenoid quinones with multiple
                                                                                                                                           Typical menaquinone molecule species have been widely researched and provide an extremely effective method for
                     similar structures.
            Observation of
                                                                                                                                           classification and identification at the genus level. This composition nomenclature is essential for naming a new
                                                                                                                                           species. LC/MS is a powerful tool for the identification and determination of the composition ratio of menaquinone
          Microorganism Species
                                                                                                                                           molecule species.


                        Features of the LCMS-2020 Ultra Fast Liquid Chromatograph Mass Spectrometer                                                                        MK-n (Hm)         n: number of isoprene units
                                                                                                                                                                           ············· Expression (1)   m: number of hydrogen atoms related to saturation of the isoprene side chains
                         Rapid 15 msec positive/negative ion switching time permits high-speed detection (MS measurements) that follows
                         high-speed LC separation.                                                                                                                                                                                                     Microorganism Species  Identification of
                         The newly developed Qarray ion optical system achieves superior sensitivity, reproducibility, and linearity.     Fig. 1 Chemical Structural Formula of Menaquinone
                         Rapid, 15,000 u/sec scanning achieves high ion permeability while maintaining resolution.
            Identification of
                         The LCMS-2020 dramatically enhances long-term stability and ease of maintenance.                                  Test and research flow
                         LCMSsolution maximizes analysis performance through comprehensive functions including data comparisons, peak
          Microorganism Species
                                                                                                                                           1. Pulverize freeze-dried bacteria (100 to 300 mg) and extract the lipid components using chloroform/methanol (2:1).
                         integration, and report output.
                                                                                                                                           2. Re-dissolve the filtrate in acetone, and roughly separate the menaquinones using TLC silica gel.
                                                                                                                                           3. Re-extract and re-concentrate the menaquinones and perform LC/MS analysis (in ESI-positive mode).

                                                                                                                                                                                                 Analytical Conditions
                                                                                                                                             Int.  TIC                                           Column      : Shim-pack FC-ODS
                                                                                                                                                717.00
                                                                                                                                                719.00                                           Mobile phase  : acetonitrile / methanol /
                                                                                                                                                721.00               MK-8(H8)                                 10 mM ammonium acetate = 2 / 6 / 15
                                                                                                                                             2000e3 723.00        MK-8(H6)                                                                             Specific Microorganisms  Detection of
                                                                                                                                                725.00                                           Flow rate   : 0.2 mL/min
                                                                                                                                                727.00
                                                                                                                                                729.00                                           Column temp.  : 40 °C
                                                                                                                                             1500e3 731.00
                                                                                                                                                733.00                                           Injection volume  : 6 µL
                                                                                                                                                735.00                                           Probe voltage  : 4.5 kV (ESI-positive mode)
            Detection of
                                                                                                                                             1000e3
                                                                                                                                                                                                 CDL temp.   : 300 °C
                                                                                                                                             500e3                                               Nebulizing gas flow : 1.5 L/min
                                                                                                                                                                                                 Drying gas pressure : 0.15 MPa
          Specific Microorganisms
                                                                                                                                              0e3                                                CDL voltage  : 25 V
                                                                                                                                                     5.0  10.0  15.0  20.0  25.0  30.0  35.0  min  Qarray DC voltage  : Scan mode
                                                                                                                                             Int.  TIC
                                                                                                                                                785.00                                           Qarray RF voltage  : Scan mode
                                                                                                                                                787.00                         MK-9(H8)
                                                                                                                                                789.00                    MK-9(H6)               Scan range  : m/z 200-400 (1.0 sec/scan)
                                                                                                                                                791.00
                                                                                                                                              7.5e6 793.00                                       SIM         : m/z 331.1, 329.1, 315.1, 313.0 (0.25 sec/ch)
                                                                                                                                                795.00                  MK-9(H4)       MK-9(H10)
                                                                                                                                                797.00
                                                                                                                                                799.00                                 MK-9(H12)
                                                                                                                                                781.00
                                                                                                                                              5.0e6 783.00
                                                                                                                                                                                                 Specific menaquinone molecule species are
                                                                                                                                              2.5e6
                                                                                                                                                                                                 detected for each actinomycete (Fig. 2 and Table 1).
                                                      LCMS-2020                                                                               0.0e6  5.0  10.0  15.0  20.0  25.0  30.0  35.0  min  Analysis based on such a chemical classification    Components  Analysis of Microorganism-Derived
                                                      Ultra Fast Liquid Chromatograph Mass Spectrometer                                                                                          index is important for the classification and
                                                                                                                                          Fig. 2 Total Ion Chromatogram and Mass Chromatogram of Menaquinones  identification of bacteria. LC/MS is an essential
          Components
                                                                                                                                                                                                 analysis tool for this purpose.
            Analysis of Microorganism-Derived
                                                                                                                                             Table 1 Menaquinone Molecule Species Content and Relative Retention Time based on MK-6 (H0) for Typical Actinomycete Species
                                                                                                                                                              Actinomycetes          MK-7  MK-8  MK-9              MK-10
                                                                                                                                                                                JCM No.  H0  H0  H2  H0  H2  H4  H6  H8  H0  H2  H4  H6
                                                                                                                                                       Thermoactinomyces candidus  3180T  1.00  1.50
                                                                                                                                                       Rhodococcus equi         1311T         1.77
                                                                                                                                                       Amycolata autotrpphica   4348T         2.05
                                                                                                                                                       Nocardia asteroides      6043T            1.86                                                  Other Applications
                                                                                                                                                       Tsukuamurella pauromethabolum  3226T      1.87  2.11  2.38
                                                                                                                                                       Streptosporangium vulgare  3028T                  2.47
                                                                                                                                                       Amycolatopsis orientalis sub sp. Orientalis  4600T  2.39  2.70  3.07
                                                                                                                                                       Actinomadura madurea     7436T                       2.66  3.04
          Other Applications
                                       Browser screen                         Analytical conditions optimization screens                               Streptomyces albus subsp. Albus  4450T               2.67  3.05
                                                                                                                                                       Kitasatosporia setae     3304T                              2.53  2.91  3.36  3.81
                                                                LCMSsolution Ver.5                                                                     Nocardiopsis assonvillei subsp. Dassonvillei  7437T  2.39           3.32
                                                                LCMS-2020 Workstation                                                                  Saccharothrix australiensis  3370T
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