Name: standardized identification of compound structure in tibetan medicine using ion trap mass spectrometry and multiple-stage fragmentation analysis
Uploaded: 2023-03-17T14:10:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis at JoVE.com

This work was funded by the Xinglin Talent Program of Chengdu University of TCM (No. 030058191), the Nature Science Foundation of Sichuan (2022NSFSC1470), and the National Natural Science Foundation of China (82204765).

1. Sample preparation
<ol>
	<li>Accurately weigh 1 g of the AMS sample, and place it in a conical flask with 30 mL of 80% methanol. Transfer the mixture to an ultrasound bath sonicator for 30 min of extraction at 25 &#176;C. Centrifuge the sample at 14,000 x g for 5 min. 
	NOTE: The frequency of the ultrasound bath sonicator is 40 KHz.</li>
	<li>Prepare an injection syringe and a microporous membrane filter (0.22 &#956;m, organic only). Filter the supernatant into a 2 mL sample bottle.</li>
</ol>
2. MS setting
<ol>
	<li>Turn on the switch of the vacuum pump. Open the main valve of the argon cylinder and the partial pressure valve, and adjust the pressure to approximately 0.3 MPa. Open the nitrogen valve. 
	NOTE: Wait for at least for 8 h to ensure a sufficient vacuum degree for the experimental conditions. Check that the gas pressure of argon and nitrogen is high enough before analysis.</li>
	<li>Launch the MS control software. Click on Heated SEI Source in the software panel, and enter the MS parameters, including the heater temperature (350 &#176;C), sheath gas flow rate (35 arb), aux gas flow rate (15 arb), spray voltage (3.8 KV for positive mode, &#8722;2.5 KV for negative mode), and capillary temperature (275 &#176;C). Click on the Apply button to activate the ion source.</li>
</ol>
3. LC prerun, method establishment, and MS acquisition
<ol>
	<li>Prepare mobile phase A and mobile phase B using 0.1% formic acid aqueous solution and pure acetonitrile, respectively. Degas them in an ultrasound bath sonicator for at least 15 min. Connect the solutions to the A and B fluid passages, respectively (Figure 1A). Prepare a methanol-water (1:9 v/v) solution, and then fill it into the cleanout fluid bottles of the pump and injector by hand. 
	NOTE: The frequency of the ultrasound bath sonicator is 40 KHz.</li>
	<li>Launch the LC-MS control software.
	<ol>
		<li>Click on the Direct Control button to open the LC control panel. Open the purge valve in the counterclockwise direction on the pump module (Figure 1B).</li>
		<li>Click on the More Option button to open the pump setting, and set the purge parameters at 5 mLmin&#8722;1 for 3 min. Click on the Purge button to start the bubble removal. Subsequently, close the purge valve.</li>
	</ol>
	</li>
	<li>Click on the Prime Syringe, Wash Buffer Loop, and Wash Needle Externally buttons to rinse the syringe for three cycles, the loop for one cycle, and the needle for one cycle, respectively. Place the sample bottle in the sampler (Figure 1C).</li>
	<li>Click on the Instrument Setup button to open the method-editing window. Click on the New button to create a new LC-MS instrument method.</li>
	<li>Establish a total run time for the LC method. Next, enter values to set the pressure limit, total flow rate, flow gradient, sample temperature, column temperature, and ready temperature delta in the method-editing window. 
	NOTE: The default total flow rate of the mobile phase is constant at 0.3 mL/min with 50% A and 50% B and without column temperature in the absence of a chromatographic column. The default values of sample temperature and ready temperature delta are 15 &#176;C and 0.1 &#176;C, respectively. Other settings depend on the type of liquid chromatography column used.</li>
	<li>Select the General MS or MSn experiment type for the MS method. Enter values to configure the acquisition time, polarity, mass range, divert value number, and divert value duration. Click on the Save button to configure the settings as an instrument method. 
	NOTE: The default settings without a chromatography column are as follows: acquisition time, 2 min; polarity, positive or negative; mass range, 100 to 1,200; divert value number, 2; and divert value duration, 1.99 min.</li>
</ol>
4. Operating multiple-stage mass spectrometry
<ol>
	<li>Click on the Sequence Setup button to open the sequence table.
	<ol>
		<li>In the table, enter the following information: sample type, file name, path, sample ID, instrument method, position, and injection volume.</li>
		<li>Click on the Save button to record the sequence table, and then click on the Start Analysis button to implement the settings and initiate the MS acquisition. 
		NOTE: The default sample type is selected as unknown. The instrument method is the method saved in step 3.6. The sample bottle is placed in its unique location in the sample room. For example, RA1 is the first location in the first row of the red area in the sample room. The default injection volume is usually 2 &#956;L, which depends on the concentration of the sample.</li>
	</ol>
	</li>
	<li>Double-click on the raw file in explorer to load the MS data into the data processing software. In the base peak chromatogram (BPI), select the area with the maximum area under the curve (AUC) by clicking and dragging the mouse. The corresponding MS spectra will be displayed in the same window.</li>
	<li>Select a targeted ion for the next MS/MS analysis.
	<ol>
		<li>Reopen the method-editing window. In the MSn Setting table, set the m/z of the targeted ion to one decimal place in the Parent Mass column.</li>
		<li>Select Collision mode, and enter the collision energy (CE) value. Set the MS/MS scan range. Click on the Save button to record the MS method, and enter a new file name in the sequence table. Click on the Start button to initiate the MS/MS acquisition. 
		NOTE: The MS/MS scan range was 40%-130% of the targeted parent ion. The default CE value in collision-induced dissociation (CID) mode is 35%.</li>
	</ol>
	</li>
	<li>Double-click on the raw file in explorer to load the MS/MS raw file into the data processing software.
	<ol>
		<li>Identify the strongest fragment ion in the MS/MS spectrum, and enter its m/z value into the MSn method list. In the MSn Setting table, set the MS3 parameters, including collision mode, CE value, and scan range.</li>
		<li>Click on the Save button to record the MS method, and enter a new file name in the sequence table. Click on the Start button to initiate the MS3 acquisition.</li>
	</ol>
	</li>
	<li>Double-click on the raw file in explorer to load the MS3 raw file into the data processing software. Repeat step 4.4 to obtain the MS4 spectrum.</li>
	<li>Complete the MSn experiment when no stable fragment ions are observed in the spectrum.</li>
</ol>
5. Manual MSn data analysis
<ol>
	<li>Double-click on the raw files to open all the mass spectra from MS to MSn. Manually calculate the m/z difference values between the ion and the corresponding fragment ions. 
	NOTE: For example, the m/z difference value between the ion (m/z 617.25) and the corresponding fragment ions (m/z 571.28) was 45.97 in MS/MS, the m/z difference value between the ion (m/z 571.28) and corresponding fragment ions (m/z 525.38) was 45.90 in MS3, and the m/z difference values between the ion (m/z 525.38) and the corresponding fragment ions (m/z 344.93 and 273.16) were 180.45 and 252.22 in MS4, respectively.</li>
	<li>Manually draw the &#34;core&#34; structure according to MS4 results (the last level of MSn). Manually derive the original structure using functional groups or molecular segments based on the m/z difference value. Manually draw the molecular cleavage paths according to each molecular structure in MSn. Examples of manual molecular derivation are detailed in the representative results section.</li>
</ol>

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The authors declare no competing financial interests.

IT-MS and its MSn technology offer a new approach to identifying the structure of trace TCM compounds. Unlike Q-TOF-MS, which could not deeply identify the fragment ions, IT-MS with MSn technology excels due to its ability to isolate and accumulate ions. This article outlines a method for identifying trace compounds in Tibetan medicine using the IT-MS and MSn technique. The method utilizes the n value in MSn to determine the amount of fragment ion information provided. The crucial steps in this method include selecting the appropriate scan range and adjusting the CE value, which lead to the identification of valuable fragments.
In general, the MSn analysis of saccharides is best performed in positive ion mode16, while phenolic acids and alkaloids are best analyzed in negative ion mode. The response of the compound in the ESI source can be improved by adjusting the mobile phase with additives such as formic acid, acetic acid, and ammonium acetate17. An atmospheric-pressure chemical ionization source can be considered for compounds with weak polarity. Choosing an appropriate scan range can increase the intensity of the fragment ions, which is beneficial for the next stage of MSn because of the inevitable energy decay in each MSn. The m/z of the fragment ion should be located in the central region of the scanning range to obtain the best corresponding intensity. If an ion has double or multiple charges, fragment ions with higher m/z values can be obtained by decreasing the charge number during fragmentation. In this case, the end m/z of the scanning range should be set to be larger. The CID mode is suitable for most compounds in MSn analysis18. If the intensity of the fragment ion is insufficient, the CE value can be increased by 5% at a time. When there are multiple, complex fragment ions in MSn, a lower CE value is needed to control the ion dissociation. The pulsed-Q collision-induced dissociation mode, which is suitable for small molecules, provides more detailed information about low-molecular weight fragment ions than CID mode19. The electron transfer dissociation (ETD) model is dominant in peptide fracture and protein identification but is rarely used to identify the TCM components20. The ETD mode can be used to investigate unknown compounds containing disulfide bonds21.
Although the MSn method has many advantages for structural identification compared to other MS techniques, there are still some limitations. First, none of the collision modes are suitable for all TCM compounds. A reasonable choice of collision mode and manual adjustment of the collision energy can improve the fragment ions. In addition, with the MSn method, it is difficult to distinguish the position of functional groups in large molecules with complex isomers. Identifying the functional group sites is a challenging task that requires experienced researchers. Manual post-analysis and long MSn data processing time are also significant barriers that discourage researchers from utilizing this technology. Q-TOF-MS is popular among researchers due to its high measurement accuracy, resolution, and ease of use with databases. However, IT-MS is a good solution for unidentified ions and trace ions due to its ability to isolate and accumulate ions and perform multiple stages of analysis. The integration of Q-TOF and IT-MS could provide a optimal solution for the full qualitative analysis of TCM samples. MSn technology is widely used in fields such as food, environmental science, and medicine, and its popularity and use in various fields are expected to increase with the improvement of IT-MS instrumentation.

The qualitative analysis of trace components in traditional Chinese medicine (TCM) has become a crucial topic in research. Due to the high numbers of compounds in TCM, it is difficult to isolate them for nuclear magnetic resonance spectrometer (NMR) or X-ray diffractometer (XRD) analysis, making mass spectrometry (MS)-based methods that only require low sample volumes increasingly popular. Additionally, liquid chromatography (LC) coupled with MS has been widely used in TCM research in recent years for the improved separation of complex samples and qualitative analysis of chemical compounds1. One common method is liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS), which is widely used in qualitative research on Tibetan medicine2. With this method, complex components are enriched and separated in an LC column, and the mass-to-charge ratio (m/z) of the adduct ions is observed using an MS detector. Searching tandem MS (MS/MS or MS2) databases is currently the fastest approach for confident compound annotations in small molecule analysis using quadrupole time-of-flight (Q-TOF) MS and Orbitrap MS3. However, the poor quality of databases and the presence of various isomers hinder the identification of unknown compounds. In addition, the information provided by the MS/MS database is limited4,5,6,7. It is significant to investigate the chemical compounds in each TCM using a general protocol that can be widely applied to other TCM.
IT-MS captures a wide range of ions by applying different radio frequency (RF) voltages to the ring electrodes8. IT-MS can perform time-series multiple-stage MS scans in diverse chronological orders, providing ingredient multiple-stage MS (MSn) fragmentation, where n is the number of product ion stages9. Linear IT-MS is considered the best for structure identification as it can be used for sequential MSn experiments10. Targeted ions can be isolated and accumulated in linear IT-MS1. The MSn (n &#8805; 3) in IT-MS provides more fragment information than MS/MS in Q-TOF-MS. Since IT-MS cannot lock the target ion and its fragment ions, it is a powerful tool for the structure elucidation of unknown compounds, including isomers1. MSn technology has been widely applied to the structural analysis of unknown proteins, peptides, and polysaccharides11,12. The abundance level of fragment ions in MSn provides more molecular fragment information on targeted compounds in complex samples than MS/MS in Q-TOF-MS. Hence, applying MSn technology to structural identification in TCM is essential.
Tibetan medicine is a significant component of TCM13, and these medicines are primarily derived from animals, plants, and minerals found in the plateau area14. The Tibetan medicine Abelmoschus manihot seeds (AMS) is the seed of Abelmoschus manihot (linn.) medicus. AMS is a traditional herbal medicine used to treat conditions such as atopic dermatitis, rheumatism, and leprosy. It contains chalcone, which possesses antibacterial, antifungal, anticancer, antioxidative, and anti-inflammatory effects15. In the present study, MSn procedures were improved, and a detailed method was developed to identify compound structures in the Tibetan medicine AMS using IT-MS and MSn. Certain MS parameters, including the ion mode, scanning range, and collision mode, were optimized to overcome problems in identifying trace compounds. This study aims to promote the standardized structure identification of trace compounds in TCM.

<table><tbody><tr><td>Acetonitrile</td><td>Thermo Scientific</td><td>CAS 75-05-8</td><td>LC-MS grade</td></tr><tr><td>Formic Acid</td><td>Knowles</td><td>CAS 64-18-6</td><td>HPLC grade</td></tr><tr><td>Linear ion trap mass spectrometer</td><td>Thermo Scientific</td><td>LTQ XL</td><td></td></tr><tr><td>liquid chromatograph</td><td>Thermo Scientific</td><td>U3000</td><td></td></tr><tr><td>LTQ Tune</td><td>Thermo Scientific</td><td>version 2.8.0</td><td>MS control software</td></tr><tr><td>Methanol</td><td>Thermo Scientific</td><td>CAS 67-56-1</td><td>LC-MS grade</td></tr><tr><td>Pure water</td><td>Thermo Scientific</td><td>CAS 7732-18-5</td><td>LC-MS grade</td></tr><tr><td>Xcalibur</td><td>Thermo Scientific</td><td>version 2.0</td><td>LC-IT-MS operational software</td></tr></tbody></table>

standardized identification of compound structure in tibetan medicine using ion trap mass spectrometry and multiple-stage fragmentation analysis

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Cellobiose was used as a model to verify the feasibility of MSn in positive ion mode. As shown in Figure 2A, the ESI-MS (positive ion mode) of cellobiose [C12H22O11]+ produced the protonated molecule [M+H]+ at m/z 365. The product ion scan (CID-MS/MS) of [M+H]+ at m/z 365 resulted in the second fragment ion at m/z 305 (Figure 2B), which was further analyzed using MS3 and MS4 analyses (Figure 2C,D). MS3 analysis resulted in the third fragment ion at m/z 254, and the MS4 analysis resulted in the fourth fragment ion at m/z 185. The MS/MS analysis (Figure 2E) revealed that the lost fragment ion at m/z 60 indicated a sequence of ion fragmentation at m/z 365, namely ring-opening hydrolysis (marked in blue), C-C bond cleavage (marked in red), and dehydration (marked in green). Similarly, the MS3 analysis revealed that the lost fragment ion at m/z 60 indicated the C-C bond cleavage (marked in red) of an ion at m/z 305. The MS4 analysis showed that the lost fragment ion at m/z 60 implied hydrolysis (marked in blue) and dehydration (marked in green), resulting in the cleavage of the ion with m/z 245 into an ion with m/z 185. The step fracture in the MSn analysis indicated that this method was feasible for investigating the structure of carbohydrates.
The preliminary qualitative analysis of AMS using LC-Q-TOF-MS revealed the presence of numerous unknown compounds. One of these, an ion at m/z 617, was selected for MSn analysis in negative mode. The product ion scan (CID-MS/MS) of the [M-H]&#8722; at m/z 617 in AMS produced a second fragment ion at m/z 571. The MS3 analysis of this fragment ion produced a third fragment ion at m/z 525, and the MS4 analysis produced fourth fragment ions at m/z 345 and 273 (Figure 3A-D). The MS3 of m/z 571 afforded a fragment ion at m/z 525 by the loss of the CH2OH portion as methanol (&#8722;32 Da) and the OH portion (&#8722;18 Da) as water. These MS4 results were used for the manual identification of the &#34;core&#34; structure of the compound, and its original structure was determined by comparing the m/z values of the ion and its fragment ions. The molecular structure of the compound at m/z 617 and its cleavage paths in MSn are shown in Figure 3E. Another unknown compound at m/z 365 was analyzed in positive mode using MSn. The product ion scan (CID-MS/MS) of the [M+H]+ ion at m/z 365 in AMS produced second fragment ions at m/z 299, m/z 329, and m/z 347. The MS3 analysis of these fragment ions produced a third fragment ion at m/z 231 (Figure 4A-C). The molecular structure and cleavage mechanism of the compound at m/z 365 are shown in Figure 4E.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65054/65054fig01.jpg" /> 
Figure 1: Identifying unknown compound structures in Tibetan medicine using IT-MS and multiple-stage mass spectrometry analysis. (A) The mobile phase forliquid chromatography. (B) The liquid chromatography pump. (C) The sample room. (D) The ion source for MS. (E) The internal structure of the ion trap module in MS. (F) The MS4 spectrum. (G) The molecular structure information from the MS4 results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65054/65054fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65054/65054fig02.jpg" /> 
Figure 2: Multiple-stage fragmentation of cellobiose via IT-MS in positive ion mode. (A) Original mass spectrum of cellobiose. (B) Fragment ions in the MS/MS spectrum. (C) Fragment ions in the MS3 spectrum. (D) Fragment ions in the MS4 spectrum. (E) The cleavage mechanism and molecular structure of cellobiose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65054/65054fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65054/65054fig03.jpg" /> 
Figure 3: Multiple-stage fragmentation and structural analysis of the unknown AMS compound ion at m/z 617 via IT-MS in negative ion mode. (A) Partial mass spectrum of AMS. (B) Fragment ions in the MS/MS spectrum. (C) Fragment ions in the MS3 spectrum. (D) Fragment ions in the MS4 spectrum. (E) The cleavage mechanism and molecular structure of the AMS compound ion at m/z 617. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65054/65054fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65054/65054fig04.jpg" /> 
Figure 4: Multiple-stage fragmentation structural analysis of the unknown AMS compound ion at m/z 365 via IT-MS in positive ion mode. (A) Partial mass spectrum of AMS. (B) Fragment ions in the MS/MS spectrum. (C) Fragment ions in the MS3 spectrum. (D) The cleavage mechanism and molecular structure of the AMS compound ion at m/z 365. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65054/65054fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis at JoVE.com

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

standardized-identification-compound-structure-tibetan-medicine-using

Nanyang Technological University

Research

JoVE Journal

Chemistry

816 Views.  Chengdu University of Traditional Chinese Medicine. Deze methode kan onderzoekers helpen bij het analyseren van de structuren van Tibetaanse medicijnverbindingen, met name de componenten van de boom. Het kan inzicht geven in de kwalitatieve studie van het gehalte van verbindingen in de natuurlijke geneeskunde. Het belangrijkste voordeel van deze techniek is dat de nauwkeurigheid van de samengestelde structuur die in dit geval wordt verkregen, hoger is dan die van de database.Deze techniek kan worden gebruikt bij de structuuranalyse van ...

Video: Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

Analyzing Tibetan Medicinal Plant APB Using 2D-HPLC-MS with Molecular Networking

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate ...

Medicine

Tandem Mass Spectrometry

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help elucidate its composition and sequence. This is accomplished by having mass spectrometers in series. The first spectrometer ionizes a sample and filter ions of a specific mass to charge ratio. Filtered ions are then fragmented and passed to a second mass spectrometer where the fragments are analyzed.
This video introduces the principles of tandem mass spectrometry, including mass-to-ratio selection and dissociation methods. Also shown is a general procedure for analyzing a biochemical compound using tandem mass spectrometry with collision-induced dissociation. The applications section covers selection reaction monitoring, determination of protein post-translation modifications, and detection of tacrolimus levels in blood.
<div class="chapter_text" id="script_0">
Tandem mass spectrometry links together multiple stages of mass spectrometry to first isolate a biomolecule, and then determine aspects of its chemical makeup. Biomolecules have large, complex structures, making it difficult to determine their molecular composition. Tandem mass spectrometry selects a molecule of interest that is later fragmented into multiple subunits, which can help elucidate its identification and sequence. This video will show the concepts of tandem mass spectrometry, a general procedure, and some of its uses in biochemistry.
</div>
<div class="chapter_text" id="script_1">
Tandem mass spectrometry begins as a typical mass spec instrument: with an ion source, which converts the sample into ions, and a mass analyzer, which separates the ions based on their mass-to-charge ratio. A common mass analyzer, the quadrupole, only allows ions with a specific ratio through, while the others crash into the rods of the apparatus. The species allowed through, called the precursor ion, is the biomolecule of interest. The ion moves into a collision cell, typically another quadrupole, where energy is applied to fragment the ion in a predictable pattern.
These fragments move into another mass analyzer, such as a time-of-flight, which separates these &#34;product ions&#34;. The product ions are then sent to the detector, as in a normal MS instrument. In the case of an unknown protein, the resulting spectrum contains numerous overlapping fragments, making a definitive complete sequence of the biomolecule difficult to generate. However, the spectral pattern is unique for a given protein. Analysis software compares the spectrum to a database of known peptide sequences, elucidating the unknown protein from the overlapping fragments.
Depending on the sample and desired degree of fragmentation, multiple fragmentation methods are possible. Fragmentation patterns depend on how the energy is transferred, its amount, and how it is distributed through the precursor ion. Energy can be transferred via neutral particles, radiation, or electrons. Using neutral atoms, a process called collision-induced dissociation or CID, primarily cleaves at the peptide bond between the amino acids, ideal for their identification.
Now that the basics of the technique have been covered, let&#39;s look at CID tandem mass spectrometry being used to study a component of bacterial cell envelopes.
</div>
<div class="chapter_text" id="script_2">
As with all mass spectrometric experiments, the first step is to ionize the sample. For biomolecules, this is typically done with matrix assisted laser desorption or electrospray ionization. The precursor ion signal is then optimized by tuning of the ion optics. Once done, the target is isolated and the fragmentation method is chosen, such as CID.
The strength of an applied voltage, which accelerates the precursor ion into the collision cell, affects the degree of fragmentation. This voltage is increased until the precursor is roughly 10% abundance compared to the highest product ion. Multiple spectra are acquired and averaged until a sufficient signal-to-noise ratio is achieved. The number of scans needed is dependent on the signal intensity of the original precursor ion and can range from 3 to 300.
The analyte in this example, lipid A from Escherichia coli K-12, had 19 major fragments after CID. Lipid A&#39;s general structure is well known, allowing software to reconstruct the specific composition from the sample.
</div>
<div class="chapter_text" id="script_3">
Now that we&#39;ve looked the procedure, let&#39;s look at some of the ways tandem mass spectrometry is used in biochemistry.
A common scanning mode in tandem mass spectrometry is selected reaction monitoring, or SRM. In SRM, both mass analyzers are fixed to a selected mass-to-charge ratio, focusing on specific precursor and product ions. Because of SRM&#39;s high degree of sensitivity, the spectra of peptide standards of known concentration can be utilized and compared to that of the unknown samples, allowing proteins of interest to be quantified.
Proteins are commonly modified after translation, typically by the addition of functional groups such as methyl groups, phosphate groups, or sugars, known as glycans. These are important in cell signaling processes, elucidating how cells communicate with one another. Because tandem mass spectrometry fragments the proteins into smaller components, it is possible to determine the location of the PTM to the specific fragment or even an amino acid. Some modifications, such as acetylation and trimethylation, are difficult to differentiate by mass alone, so chromatographic separation is performed before the mass spectrometry.
Many analytes in patient&#39;s blood are found at concentrations below the limit of detection for typical mass spectrometry. Another advantage of SRM is that it discards all but one product ion, increasing the sensitivity and enhancing the lower detection limit by up to 100 fold. In this example, the immunosuppressant drug, tacrolimus, could be detected at levels of 1 ng/mL.
</div>
<div class="chapter_text" id="script_4">
You&#39;ve just watched JoVE&#39;s video on tandem mass spectrometry. This video described the theory of the instrument, went over a general procedure, and explained some of the ways the technique is currently being utilized. Thanks for watching!
</div>

Tandem Mass Spectrometry: Principle, Instrumentation, Uses

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help ...

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

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