This manuscript has been authored by Battelle Memorial Institute under Contract No. DE-AC05-76RL01830 with the U.S. Department of Energy. The U.S Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for U.S. Government purposes.

1. Sample Preparation
<ol>
	<li>Generate a mixed aqueous/organic product stream via continuous flow HTL of algae according to the reactor design and experimental procedure found in the literature10,11.</li>
	<li>Use a gravity separator to separate the product stream into an aqueous phase and organic phase.</li>
	<li>Filter 10 ml of the HTL aqueous phase using a 0.45 &#181;m syringe filter and store in a refrigerator maintained at 4 &#176;C for GC &#215; GC&#8211;TOF-MS analysis.</li>
</ol>
2. Instrument Components
<ol>
	<li>Use a gas chromatograph (GC) equipped with a quad-jet dual stage cooling-based modulator and time-of-flight (TOF) mass spectrometer (MS) for these experiments.</li>
	<li>Configure the auto-sampler to inject 1 &#181;l of each sample or standard into the GC. Use a randomized block design of sample and standard injections for the auto-sampler sequence as described in the literature13. The randomized block design is commonly used in quantitative studies to control for instrument operation. Our laboratory utilizes the design routinely even in comparative studies to verify instrument operation.</li>
	<li>Connect the primary and secondary column using a press-tight connector before the modulator. Ensure that both edges of both primary and secondary columns are cut straight without sharp edges before connecting to the press-tight connector.</li>
	<li>Place ferrule on the GC column and then connect primary column to the GC injector so that 5 mm of column is inside the injector.</li>
	<li>Ensure that glass liner, non-stick liner O-ring and septa for GC injector are new and free of contamination.</li>
	<li>Use 1/16 x 0.5 mm ID transfer line ferrules to connect the secondary column and transfer line. Place a 0.2 m portion of the secondary column in the transfer line.</li>
	<li>Ensure that a 0.1 m portion of the secondary column is in the modulator.</li>
	<li>Use ultrahigh purity helium gas as carrier gas for GC at a flow rate of 1.5 ml min-1.</li>
	<li>Ensure there is sufficient liquid nitrogen in the Dewar which acts as a coolant in the modulator. The level of the liquid nitrogen in the Dewar can be predicted using a pressure gauge attached to its outlet. A 69 kPa reading of the pressure gauge indicates that the Dewar is full, while 0 kPa indicates that it is empty.</li>
</ol>
3. Protocols Before Analyzing Samples
<ol>
	<li>Ensure there are no major leaks in the instrument. If the vacuum gauge reading of the TOF-MS is higher than 2.7 &#215; 10-5 Pa for 1.5 ml min-1 GC column flow rate, this indicates a major leak in the system.</li>
	<li>Set-up the quality control (QC) method and run in-built &#39;acquisition system adjustments&#39; protocol to achieve maximum signal response using manufacturer&#39;s protocol.</li>
	<li>Run in-built &#39;instrument optimization&#39; protocols of QC method, in series - filament focus, ion optic focus and mass calibration tests using manufacturer&#39;s protocol. Ensure that mass calibration test passes. This QC method ensures that all the hardware parameters of the instrument are at optimum level.</li>
	<li>Perform a &#34;leak check&#34; using manufacturer&#39;s protocol. Analyze automatically generates leak check report. Ensure that the relative concentration of 28 (N2), 32 (O2) and 18 (moisture) ions must be below less than 10%, 3% and 5% of internal standard mass spectra of 69 ion, respectively.</li>
	<li>Tune the TOF-MS using manufacturer&#39;s protocol.</li>
	<li>Run quality control method as well as TOF-MS tune protocol before and after leak check and also while analyzing samples and standards.</li>
</ol>
4. Protocol to Determine the Optimum Modulation Period of Modulator
<ol>
	<li>Arbitrarily select a long modulation period (e.g. 10 sec or 13 sec). Inject a sample as described in 2.2.</li>
	<li>Identify the retention time in second dimension of the contour plot after which no peaks elutes. Select identified second dimension retention time as optimum modulation period. Figure 2 clearly elucidate the identification of retention time in second dimension of the contour plot.</li>
	<li>Increase the modulation period used in step 4.1 and perform the analysis again if &#34;wrap around&#34; is observed18. Wrap around phenomena occurs if the peaks in the second dimension elutes below the baseline of first dimension. Example contour plot for &#39;wraparound&#39; is shown in supplementary information Figure 3.</li>
	<li>Repeat steps 4.2 and 4.3 until optimum value is determined.</li>
</ol>
5. Experimental Parameters of Instrument Set-up
<ol>
	<li>Install a polar (60 m x 0.25 mm x 0.5 &#181;m film thickness) capillary column as the primary column and a non-polar (2.3 m x 0.25 mm x 0.5 &#181;m film thickness) capillary column as the secondary column. Bake both the primary and secondary column for at least 2 hr to remove trace amounts of moisture, air and contaminants associated with new GC columns.</li>
	<li>Use ultrahigh purity helium gas as carrier gas for GC at a flow rate of 1.5 ml min-1.</li>
	<li>Set the GC injector to a temperature of 260 &#176;C and a split ratio of 1:250.</li>
	<li>Employ the following temperature program for the primary column: a constant temperature of 40 &#176;C for 0.2 min followed by a temperature ramp to 260 &#176;C at 5 &#176;C min-1, followed by a constant temperature of 260 &#176;C for 5 min.</li>
	<li>Maintain the modulator temperature 5 &#176;C higher than that of the secondary column and the secondary column temperature at 5 &#176;C higher than that of the primary column.</li>
	<li>Use an optimum modulation period of 4 sec with 0.8 sec of hot pulse and 1.2 sec of cold pulse. This value is determined based on the protocol described in section 4.</li>
	<li>Set transfer line temperature to 270 &#176;C.</li>
	<li>Set the acquisition delay or solvent delay to 0 sec.</li>
	<li>Set the lower and higher range of m/z as 35 and 800, respectively.</li>
	<li>Set the MS detector acquisition rate at 400 spectra/sec.</li>
	<li>Maintain the MS detector voltage at 150 V higher than the optimized value.</li>
	<li>Maintain the MS ion source temperature at 225 &#176;C.</li>
</ol>
6. Data Analysis
<ol>
	<li>Perform data processing using the software supplied by the instrument manufacturer.</li>
	<li>Select the following tasks in the data analysis method - Compute baseline, find peaks above the baseline, library search and calculate are/height.</li>
	<li>Track the baseline through the data file. Enter baseline offset as 0.5. 
	Enter expected peak width of 15 sec in the first dimension and 0.15 sec in the second dimension.</li>
	<li>Set signal-to-noise ratio as 5,000 and similarity values of &#62;850 for identification of compounds.</li>
	<li>Select a commercially available mass spectral library to identify chemical compounds present in samples and set the library search mode to forward.</li>
	<li>Process the data files using this data analysis method using manufacturer&#39;s protocol. It requires at least 1 hr to process a data file.</li>
</ol>

The authors declare that they have no competing financial interests.

Results clearly illustrate the ability of the column combination of polar &#215; non-polar to resolve polar compounds and light volatiles present in the aqueous fraction of algae bio-crude without prior sample preparation techniques. Drastic peak tailing was observed for organic acids and N-compounds while using the non-polar&#215; polar column combination. This peak tailing was not observed for the early eluting light organics. This behavior has been reproducible when verifying the instrument is free of leaks (the vacuu...

Steady growth in demand for liquid fuels, finite fossil fuel resources, uncertainty of fossil fuel supplies, and concerns over the increasing concentration of greenhouse gases in the atmosphere have increased global awareness for renewable resources1. Solar energy (including photovoltaics and solar-thermal), wind energy, hydropower, geothermal, and biomass are the primary renewable sources that could potentially replace fossil-derived energy2. Of these, biomass is the only carbon-based alternative energy resource for the production of liquid transportation fuels and high-value chemicals3. Biomass includes any organic material such as forest resources, agricultural residue, algae, oilseeds, municipal solid waste, and carbon-rich industrial wastes (e.g. from pulp and paper industry or from food processing)1. Biomass is classified into two broad categories: lignocellulosic and non-ligneous feedstocks based on compositional characteristics. Lignocellulosic biomass consists of carbohydrates and lignin, while non-ligneous feedstocks have proteins, carbohydrates and lipids/oils4. Lignocellulosic feedstocks, derived from terrestrial plants, can only satisfy 30% of the current liquid fuel (gasoline, jet fuel, and diesel) demand if sustainably cultivated and harvested5,6. Hence, non-ligneous aquatic microorganisms, such as microalgae and fungi, are considered potential feedstocks for the production of renewable liquid fuels to complement lignocellulosic resources.
Microalgae feedstocks have the potential to satisfy current liquid transportation fuels demand7,8. Algae have many advantages: high areal productivity8, the ability to grow in low-quality, brackish, or sea water9, and the ability to accumulate energy-dense triglycerides or hydrocarbons7,8. Hydrothermal liquefaction (HTL) is a viable and scalable conversion process which utilizes water naturally associated with algal or aquatic feedstocks10,11. It is a thermo-chemical process with operating temperatures of 250-400 &#176;C and operating pressures of 10-25 MPa which produces a liquid product, or bio-crude, which can be upgraded into a fuel blend stock. Bio-crude produced from HTL of algae has distinguishable and easily separable organic and aqueous fractions. The organic fraction of bio-crude can be efficiently converted into a refinery ready blend stock via catalytic hydro-treating processes11. The aqueous fraction of bio-crude contains ~30% of the total carbon present in the algal feedstock. Although thousands of compounds have been identified in the HTL aqueous stream, the predominant fractions consist of low molecular weight oxygenates (including acids, alcohols, ketones, and aldehydes) formed by the degradation of carbohydrates and lipids, and nitrogen heterocyclics (including pyrroles, pyridines, pyrazines, and imidazoles) derived from protein decomposition12. Studies on utilizing the aqueous fraction to improve overall process economics as well as sustainability are ongoing. Synthesis gas can be produced from the aqueous fraction of algae bio-crude via catalytic hydrothermal gasification10,13,14. Alternatively, organics in the aqueous fraction can also be catalytically converted to fuel additives and specialty chemicals. Research on optimizing catalytic hydrothermal gasification and catalyst screening studies for conversion of organics in the aqueous liquid phase is currently underway at the Pacific Northwest National Laboratory (PNNL). For this work, qualitative as well as quantitative characterization of the aqueous fraction of algae bio-crude is required. Since the aqueous fraction of algae bio-crude is considered a waste stream, there are very few studies that have analyzed the aqueous fraction of algae bio-crude13,15. Moreover, recent studies concluded that converting this HTL algae water into high-value bio-products would improve the sustainability as well as economics of an HTL-based bio-refinery11. Therefore, this study focused on developing a method for qualitative characterization of the aqueous fraction of bio-crude obtained from HTL of algae by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC &#215; GC&#8211;TOF-MS).
GC &#215; GC&#8211;TOF-MS is the most promising chromatographic analytical technique to increase resolution (or separation of chemical compounds in a sample), peak capacity (i.e. number of resolved peaks), signal-to-noise ratio (for identification of chemical compounds with high confidence), and to avoid co-elution of chemical compounds16. In order to maximize resolution, peak capacity, and signal-to noise ratio, two GC columns with different stationary phases are connected in series using a press-fit connector or micro-union17(see Figure 1 which is a block diagram of GC &#215; GC&#8211;TOF-MS system used in this study). A modulator is located between the press-fit connector and secondary columns to trap, refocus, and re-inject the effluents from the primary column into the secondary column18. Modulation occurs on the secondary column in the present study as shown in Figure 1. The secondary column is then connected to the TOF-MS via a transfer line assembly.
GC &#215; GC&#8211;TOF-MS was used previously for qualitative as well as quantitative analysis of organic samples such as crude oil16,19, gasoline, jet-fuel, diesel, bio-diesel, and the organic fraction of bio-fuel20-22 produced from thermo-chemical as well as thermo-catalytic conversion processes23,24. For characterization of these organic samples in GC &#215; GC&#8211;TOF-MS instruments, a long non-polar column was used as the primary column, while a short polar column was used as the secondary column. This conventional column configuration resolves chemical compounds based on differences in volatility over the first dimension, followed by polarity in the second dimension18. Aqueous or water samples from biological processes, food processing, and environmental wastes were also characterized using similar primary/secondary column configurations after the sample had been through preparation steps17,25-30. Sample preparation techniques such as derivatization, solid-phase extraction, and organic solvent extraction have all been utilized prior to GC &#215; GC&#8211;TOF-MS analysis17,27-29,31,32. These techniques were aimed at decreasing the polarity of compounds in the sample for analysis using a conventional column configuration33. An alternative strategy was employed in this study based on the nature of the sample (here polar organic compounds in water) utilizing the reverse primary/secondary column configuration for GC &#215; GC&#8211;TOF-MS analysis. Since the aqueous fraction of bio-crude produced from HTL has polar compounds13, a column combination of a primary polar column and a secondary non-polar column was used in the GC &#215; GC&#8211;TOF-MS without any upstream sample preparation. This primary/secondary column combination resolves chemical compounds based on differences in polarity over the first dimension, followed by volatility in the second dimension. Limited analytical methods exist in the literature for characterization of aqueous samples using two-dimensional gas chromatography without prior sample processing15.
The objective of this study was to determine the chemical compounds present in the aqueous fraction of algae bio-crude. To achieve this objective, a GC &#215; GC&#8211;TOF-MS data acquisition method was developed with a column combination of polar column (primary) &#215; non-polar (secondary). Klenn et al. (2015) suggested that increasing the length of the primary column (especially 60 m GC columns) and lowering the offset temperature of the secondary column with respect to the primary column would maximize peak capacity and resolution16-18. Therefore, a 60 m primary column and 5 &#176;C offset temperature of the secondary column with respect to the primary column were used in this study. The optimum modulation period was determined following a protocol described in this study (see section 4). The optimum ramp rate of GC column temperature was determined by a trial and error method and is similar to the value suggested in the literature16-18. To discuss the advantages of this column combination for aqueous samples, we have analyzed HTL algae water samples with the conventional column combination of non-polar &#215; polar. Operating parameters suggested in the literature were employed for analyzing the aqueous fraction of algal bio-crude with a non-polar &#215; polar column combination18.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">GC &#215; GC - TOF/MS</td>
			<td style="width:199px;">Leco</td>
			<td style="width:151px;">PEG4D11DLN15</td>
			<td style="width:268px;">Commercial Pegasus 4D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChromaTOF version 4.50&#160;</td>
			<td style="width:199px;">Leco</td>
			<td style="width:151px;"></td>
			<td style="width:268px;">Data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Rxi-5MS GC column</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">13420</td>
			<td style="width:268px;">2.3 m column was used from this column.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Stabilwax GC column</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">10626</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">HP-5 GC column</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:151px;">19091J-416</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Stabilwax GC column</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">15121</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Presstight Connector</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">20430</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">GC injector liner</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">23305.5</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">GC Injector ferrules</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:151px;">5181-3323</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Non-stick liner O-rings</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:151px;">5188-5365</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Transfer line ferrules</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">20212</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Ethanol</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">459844</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetone</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">414689</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetic acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">320099</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">2-butanone</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">360473</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Propanoic acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">402907</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Butanoic acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">19215</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pyridine</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">270970</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pyrazine</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">65693</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetamide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">695122</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">2,5-pyrrolididione</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">S9381</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">N-methylsuccinimide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">325384</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">N-(2-hydroxyethyl)succinimide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">444073</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
	</tbody>
</table>

qualitative characterization of the aqueous fraction from hydrothermal liquefaction of algae using 2d gas chromatography with time-of-flight mass spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A total ion chromatogram (TIC) obtained for the aqueous fraction of algae bio-crude analyzed with a column combination of polar &#215; non-polar is shown in Figure 4. Retention times and similarity or match factor values of compounds identified by searching against a National Institute of Standards and Technology (NIST) library are tabulated in Table 1. Oxygenates (such as cyclopenatanone, furanic compounds and dianhydromannitol) and organic acids (includ...

Watch this Scientific Journal Video about Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

qualitative-characterization-aqueous-fraction-from-hydrothermal

Research

JoVE Journal

Bioengineering

9.3K Views.  Pacific Northwest National Laboratory. A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating. 

Video: Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Permeabilization of Adhered Cells Using an Inert Gas Jet

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes a mechanical method to temporally permeabilize adherent cells using an inert gas jet that can facilitate the transfer of normally non-permeable macromolecules into cells. We believe this technique works by imparting shear forces on the plasma membrane of adherent cells, resulting in the temporary formation of micropores. Once these pores are created, the cells are then permeable to genetic material and other biomolecules. The mechanical forces involved do run the risk of permanently damaging or detaching cells from their substrate. There is, therefore, a narrow range of inert gas dynamics where the technique is effective. An inert gas jet has proven efficient at permeabilizing various adherent cell lines including HeLa, HEK293 and human abdominal aortic endothelial cells. This protocol is appropriate for the permeabilization of adherent cells both in vitro and, as we have demonstrated, in vivo, showing it may be used for research and potentially in future clinical applications. It also has the advantage of permeabilizing cells in a spatially restrictive manner, which could prove to be a valuable research tool.

This protocol describes a method for the temporary permeabilization of adherent cells using an inert gas jet. This technique facilitates the transfer of genetic material and biomolecules into adherent mammalian cells by the utilization of mechanical forces to disrupt the plasma membrane.

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.