1DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, 2Department of Soil Science, University of Wisconsin, Madison, 3Department of Soil and Water Science, University of Florida
This article is a part ofJoVE General. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Liang, C., Read, H. W., Balser, T. C. GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil. J. Vis. Exp. (63), e3767, doi:10.3791/3767 (2012).
Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using δ13C or ion fragments of these biomarkers in biogeochemical studies9, 10.
1. Sample Preparation and Acid Extraction
2. Sample Purification
4. Separation and Measurement
5. Derivative Validation
6. Representative Results
The protocol of the method mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination (Figure 1). An example of the analysis for glucosamine and muramic acid from standard stocks and from a soil sample is shown in Figure 2. Besides glucosamine and muramic acid, mannosamine and galactosamine (two isomers of glucosamine) can also be determined simultaneously using the method. Based on response factors of standards with respect to the internal standard myo-inositol, we can quantify these biomarkers in soil samples. The recovery standard has been used to qualitatively monitor the derivatization process. The schemes for formation of the aldononitrile acetate derivatized glucosamine and muramic acid are shown in Figure 3.
The proposed structures of the derivatives were determined by GC-EIMS-SIM for sensitivity enhancement, or soft ionization LC-ESI-TOF-MS8; the proposed structures of the ion fragments formed upon electron ionization were studied by comparing the ion spectra of the samples prepared with various isotope incorporations11. The Figure 4 shows the mass shift of the dominant ion m/z 187 of aldononitrile acetate derivatized glucosamine in three scenarios, prepared with non-labeled agents, D-acetic anhydrite, and U-13C-glucosamine. Other detailed information and explanations can be referred to our recent publications8, 11. This strategy could serve as a model to study derivative chemistry.
Figure 1. Measurement flow chart of the protocol for analysis of glucosamine and muramic acid in soil samples. The protocol mainly contains 4 steps: Acid digestion, Purification, Derivatization and GC determination.
Figure 2. GC-FID chromatograms of aldononitrile acetates of inositol, glucosamine, muramic acid, mannosamine, galactosamine and methylglucamine for standards and soil.
Figure 3. Schemes for formation of the aldononitrile acetate derivatized glucosamine and muramic acid. The number 1 represents nitrile reaction. The number 2 represents acetylation.
Figure 4. Mass spectra of aldononitrile acetate derivatized glucosamine associated with the dominant ion structures under EI mode prepared with (A) non-labeled agents, (B) D-acetic anhydrite, (C) U-13C-glucosamine. Star represents heavy isotope atom or isotope group. Please click here to see a larger version of this figure.
The presented GC-based method for the analysis of aldononitrile acetate derivatized glucosamine and muramic acid provides a relatively rapid method to quantify these amino sugars, extracted from soil. The derivatives are chemically stable, and can be determined in one analysis. The method is not restricted to soil samples, and can be simplified for samples from non-soil matrix.
The vacuum pump used in this method is built to be resistant to acid. We further suggest setting up a base trap to protect the pumps when evaporating 6M HCl solutions. Care must be taken during the derivatization steps to maintain rigorously anhydrous conditions, for both the reagents and glassware. The work should be conducted without delay. Glassware must be scrupulously clean, either by muffling at 550°C or by rinsing with solvent. Safety precautions should be taken for the handling of acids, and strong acid vapors during evaporation. Aminoglycoside, a series of antibiotic produced by soil-dwelling organisms, has been identified as a possible interference, as it co-elutes with glucosamine or muramic acid on DB-5 12, so a second column with a different stationary phase chemistry is recommended if GC-FID is the primary method used for quantifying these amino sugars. We recommend a confirmatory detector following GC elutes for future analysis. For unambiguous identification of trace biomarker in complex matrices, we suggest using sophisticated instrumentation, e.g. GC-MSMS with multiple reaction monitoring; for precise quantification of biomarker in the presence of interferents, we suggest doing calibration based on some dominant mass ions unique to the biomarker derivatives.
Confirmation of sugar structure identities was made by substituting 15N-hydroxylamine hydrochloride, deuterated acetic anhydride, and 13C and/or 15N labeled biomarkers for non-labeled hydroxylamine hydrochloride, acetic anhydride and biomarkers in preparation of the derivatives, and further monitoring predicted m/z shift of characteristic ions of the labeled versus the unlabeled derivatives. In specific, since each of acetate group contains 3 deuteriums and each nitrile group contains 1 15N atom, it can be predicted that ion m/z mass shift is decided by how many acetate groups and nitrile group will be presented in the structures of ion fragments. Similarly, 13C and/or 15N-labelling bacterial cells can both be used to determine how many C atoms in the fragment ion structures are originated from the glucosamine or muramic acid, or whether glucosamine-N or muramic acid-N exists in the fragment structures.
GC-EI-MS-SIM is gas chromatography followed by electron impact ionization and mass spectrometry using quadropole mass separation and selected ion monitoring; LC-ESI-TOF-MS is liquid chromatography followed by electrospray ionization and mass spectrometry using time-of-flight mass separation. We here point out the differences between these methods that are used to help determine the molecular weight and ion fragments. ESI-TOF-MS is a "soft ionization" technique in the ionization energy imparted to analyte molecules that is low enough, so that no fragmentation occurs and the signal produced is a highly precise measure of the molecular weight of the analyte. In GC-EI-MS, more energy is transferred to the analyte, and the molecule breaks apart yielding a number of charged fragments. The quadropole acts as a mass filter so that only those fragments of a specific mass/charge ratio reach the detector. The distribution of fragments, which is characteristic of the analyte, is shown in a graph called an ion spectrum, in which intensity or abundance is plotted against m/z. To increase sensitivity, we use selected ion monitoring (SIM), in which we program the MS to collect only a few specific m/z values rather than the entire mass spectrum.
We have nothing to disclose.
This work was supported by grants from DOE Great Lakes Bioenergy Research Center (DOE BER office of Science DE-FC02-07ER64494). We are grateful to Dr. Xudong Zhang and his group members for helpful technical discussions and invaluable comments on finalizing the protocol.
|Methanol (dry)||Acros Organics||AC326950010|
|Ethyl acetate||VWR international||BJGC100-4|
|Hydroxlamine hydrochloride||Fisher Scientific||H330-100|
|Acetic anhydride||Fisher Scientific||A10-100|
|Dichloromethane (Methylene chloride)||Fisher Scientific||D37-500|
|Hydrochloric acid (6M)||Fisher Scientific||S456-4|
|Hydroxylamine hydrochloride-15N||Icon services||IN5280|
|Acetic anhydride-2H (D6C4O3)||Acros Organics||AC174670050|
|D-glucose-U-13C||Cambridge Isotope Laboratories||CLM-1396-1|
|Ammonium sufate-15N||Cambridge Isotope Laboratories||NLM-713-1|
|Vacum pump||KNF Neuberger||D-79112|
|Hydrolysis flask||Fisher Scientific||06 423A|
|Derivatization microvial||Fisher Scientific||06-100E|
|LC-ESI-TOF-MS||Agilent Technologies||An Agilent 1200 series HPLC system coupled to an Agilent LC/MSD-TOF|