LC-MS - Science method

You need to do method troubleshooting to see if it is the HPLC condition or the MS condition that causes the problem. Please inject your four std that you missed to the MS (with no column) to prove that the MS setting is still good. If you see the signal at the expected response (good signal), then you can focus on the column. If your MS is good, inject the same std with column and use the highest organic mobile phase to elute the compound quickly near the solvent front to prove that your column does not affect your analyses. Let me know the results and we can go from there.

For LC-MS analysis, use about 2 grams of dried wild plant and callus samples, extracting with methanol at a ratio of 1:5 to 1:10. After soaking overnight, filter and concentrate the extracts for comparison.

Dileep Singh Good to hear you found the problem.

When I develop methods for formulations where filtration is important, I usually screen at least two types of filter materials (such as nylon and PTFE) and two different manufactures for each (say, FischerSci and MilliporeSigma). Additionally, I would keep the diameter (for example, 33 mm for all, otherwise there will be difference in binding and filtration capacity due to the surface area) and the material of construction for the housing the same (such as polypropylene; this is to avoid difference due to degradation and leeching, especially with aggressive solvents or base/acids). Whenever possible, I also use centrifugation as the reference solution to evaluate differences due to filtration. Since centrifugation is more straightforward, if I can get a clear solution where the expected recovery matches the experimental, that's my go to.

It's not unusual to see difference in results between labs when filtration is used. Some suggested reasons are listed below.

It's common to see a difference of a few percents in recovery using the same filter material from different manufacturers (especially if the diameters are also different).

If standard syringe filtration is used, that's prone to user variations:

- One point of variation for example is: I often prefer to discard the first few drops of filtrate as it may contain leechables or particles from the filter and/or syringe. But sometimes there is insufficient starting solution to sacrifice this amount.

- Another variation may be the final step of the filtration: Some people push the syringe plunger all the way in, thus trying to collect every last drop from the filter. This allows collecting of all volume and material but also may rupture the filter or create bubbles (and with that aggregates when we work with polymeric substances); it may also force out particles that would otherwise be retained by the filter.

- The total sample load( concentration and volume) and turbidity of the solution is important. If you only had one analyte in a clear solution, then that would filter more efficiently that filtering a solution that contains 10 analytes + matrix additives that is also turbid. The filter would then quickly exceed its binding capacity and may leave no pores for the analytes that would normally pass to traverse through the membrane.

Thanks, William Letter for your response.

Isoquercetin and quercetin are not the same compounds (isoquercetin is a 3-O-glucoside of quercetin). Therefore, they might produce different numbers of fragments at different collision energies.

What is likely happening in your case is that isoquercetin fragments to quercetin and glucose at the CE of 40 eV. Glucose is likely leaving as a neutral molecule which is why you do not see the corresponding peak in the MS, while m/z of 303 is most likely quercetin.

On the other hand, quercetin is an aglycone. As such, it has no glycoside bond that could be cleaved. This means that all of the collision energy will be used for the fragmentation of quercetin itself. This is why you see more fragments even on relatively lower CE.

If you increased the collision energy in the analysis of isoquercetin above 40 eV, you would most likely start to see some of the same fragments you see in the analysis of quercetin.

I could not understand. Kindly send a email at my address. alamfiaz@cuiatd.edu.pk

Thanks

Hi I am consultant . you can consult to me.

Chiranjeev Sharma If you just need a secondary detection before the mass spec detector, I would think about derivatizing the sample. If amino sugars is all there is (and other other small amines), think about derivatization with FDNB or by the AccQ-Tag platform from Waters. Both ways result in added chromophore to the amine with ability to analyze by UV or DAD, or fluorescence detection, either detection able to go down to ng/mL. High resolution is achieved on standard RP columns with TFA or FA/ACN mobile phase. Check the attach literature below and think if you need to alter any of the acids/salts to make it more LC-MS friendly.

If simple sugars are also present, consider running orthogonal (additional) method by PAD (ion chromatography) detection for the simple sugars. Alternatively, if you are open to outsourcing your samples, reach out to a contract testing lab or university with capabilities to run ion-chromatography with in-line mass spec detection. That will be able to analyze both amino acids, amino sugars, and free sugars together.

FDNB Derivatization:

AccQ-Tag Platform:

Ion-Chromatography with mass spec:

The technique of chromatography is appropriate for lipids, but someone with more experience is needed to develop and optimize the method of analysis.

When selecting compounds from an LC-MS library for molecular docking, prioritize those with favorable drug-likeness (e.g., Lipinski's Rule of Five), documented biological activity, and strong predicted binding affinities. Abundance is important but should be considered alongside structural characteristics and computational predictions.

Hello,

Getting started with OpenChrom can feel overwhelming at first, but it's a powerful tool for spectrometric data analysis. Here’s a quick guide to help you upload and work with your data:

For tutorials and guides, you might find these helpful:

If you're interested in general spectrometric analysis, the book Mass Spectrometry: Principles and Applications by de Hoffmann and Stroobant is an excellent resource to get a foundational understanding of the analysis processes and techniques.

The [M-H+114]⁻ adduct you observed in negative mode LC/MS could be due to an uncommon contamination or reagent interaction. Although TFA is not present, the 114 Da addition might be related to other substances or reagents present in your system (e.g., a plasticizer or solvent impurity). The fragment ions at m/z 113 and 69 suggest a specific structure breaking down in a predictable way. It's essential to check for possible contaminants in your solvents, equipment, or even the plant extract itself. You could also run a blank to see if the adduct is present without the sample.

Determining the exact phyto-constituents in a plant extract using LC-MS (Liquid Chromatography-Mass Spectrometry) involves several key steps:

1. Sample Preparation

2. LC-MS Setup

3. Mass Spectrometry Parameters

4. Method Development

5. Data Acquisition

6. Data Analysis

7. Validation

8. Interpretation

Additional Considerations

By carefully optimizing each of these steps, you can accurately determine the phyto-constituents present in your plant extract.

Note: If possible, avoid using any TFA in the mobile phase for the LC-MS system. It is too strong of an acid even when diluted. It will strongly bind to the instrument and column resulting in long term contamination of the system (Which is very expensive and time consuming to resolve).

Did you tried to find your data file in Software folder located in C-Drive ?

If not, then you can try to find there & directly copy your RAW .d file for further analysis.

Probable location followpath (may vary) -

C Drive - Program files - Agilent Masshunter - Data - (search or look by name of your data file which was provided during sample run)

1. The analyte may be present in quantities that are below the detection threshold. The sample may not have a enough concentration. 2. Configuring the Instruments: Temperature, mass spectrometer parameters, and flow rate are examples of improperly installed LC-MS settings.

Hi,

Although I am not sure I'm able to help you out, there are a few questions I have:

What machine did you use to generate the MS data?

Did you try to use the software provided with the machine to analyze your MS data?

What was your experimental setup, i.e. what are you comparing, how many groups of samples, how many replicates per sample, what kind of controls were included, did you use some kind of labeling to facilitate protein quantitation, etc.

I assume that peptide mixtures were analyzed?

Typically, LC-MS raw data is searched against a database to allow for qualitative protein identification. In addition, the number of peptides retrieved per protein could be somewhat indicative of its abundance, just like sequence coverage, although there are a lot of restrictions and considerations you'll need to take into account when (quantitatively) interpreting your data, especially when you're interested in low abundant proteins.

Post-translational modifications, such as glycosylation, add a whole new level of complexity to your (quantitative) analysis.

Perhaps, some additional information might enable the community to help you out (better).

For LC-MS analysis on macOS, user-friendly software options include 1- MZmine: an open-source tool for mass spectrometry data processing, 2- OpenChrom: which supports various chromatography and mass spectrometry data analyses. Both offer robust functionalities suitable for analyzing LC-MS data.

Dear Abdulwasiu Salaudeen Olawale,

the .d format is the Agile format for data files. It is a data folder in which the raw data, file header and method information are stored. You can copy, download or move this folder in the computer's Explorer. When you open the folder, you can see which individual files it contains. The export of Agilent .d data makes the measurement data set readable for other software, e.g. Excel, which cannot read the original Agilent data. To read and process Agilent .d (or other instrument data formats such as .raw from Thermo) you need the appropriate software from the manufacturer, e.g. Agilent Chemstation, or in your case probably the Mass Hunter software etc. or software that can process the manufacturer's data sets directly, e.g. Mestrenova for GC-MS data.

Data export is only necessary and useful if I want to process this data on another platform. Otherwise, I recommend sticking with the original software, as this is also suitable for the data format.

I have found a link on the internet that might be useful for you.

You can use it to perform an MS DIAL analysis

However, I take no responsibility for the function, the security of the site or its content.

Here is the link: https://www.reifycs.com/abfconverter/

Best regards and good luck

Joachim Horst

If you really want to learn practical HPLC and LC-MS operation, then start by getting a job at an industrial company that utilizes these tools in their projects (i.e. A Specialty Chemical Company. Pharmaceutical, Medical or Clinical Lab). You may find an employer willing to train you.ational expectation. Yes, vendors will offer such classes, but that does not mean they will provide the many years worth of hands-on training and knowledge needed to actually use a system. Proper operation and use of such complex analytical instruments requires hands-on experience, with a specific instrument, running many samples types over time. Do not be misled by sales people eager to take your money (at least so called "free" classes will not cost you money, but may also provide misinformation).

The classic texts [e.g. "Introduction to Modern Liquid Chromatography";

Author(s):Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan] which describe and illustrate HPLC (and some LC-MS) concepts are of great value to read and do not require formal classes. These texts are well worth studying both before and during training to learn the fundamental concepts (something many users never master). However, without an HPLC system to train on, books alone will not allow practical skills to be acquired. While the basic concepts are always the same, each brand and model of instrument are different, requiring different knowledge and experience to use. However, without an HPLC system to train on and an experienced professional chromatographer to guide you, books alone will not allow practical skills to be acquired. Initially, learning just the basics of HPLC (which are the most important areas of study, as "MS" is just one of the detection systems used) takes the average scientist 5-years of full-time training, with mentoring, using an actual complete instrument. Troubleshooting and method development skills take much longer. No short-cut is possible to replace the needed hands-on time and experience required.

If you really want to learn practical HPLC and LC-MS operation, then start by getting a job at an industrial company that utilizes these tools in their projects (i.e. A Specialty Chemical Company. Pharmacetical, Medical or Clinical Lab). You may find an employer willing to train you.

Hi there! I´ve used DETARCHI software which is indeed ver easy to use in the calculation of CCA and CCB.

You may contact the department of Chemistry from the University of Burgos, Spain to get the software.

"Hits (DB)" refers to the compounds detected in the sample that match entries in a reference database (DB). These hits are essentially the compounds that the analysis software predicts to be present in the sample by comparing the acquired mass spectra with those in the database. Whether you should not consider all the predicted compounds from the list for further studies before precising it.

Many possible answers and no web forum will provide the specific answer. HPLC systems are very complex and there are many causes for variation in results. All of these are best addressed in-person, on-site, evaluating the specific instrumentation AND the operator (as most problems are the result of poor training).

In your case, you will want to evaluate the condition of the HPLC system and MS settings. If MS peak AREAS alone are increasing (or changing), this may be the result of column fouling, detector overloading (contamination). Sample matrix may be interferring with the detection. Review sample cleanup procedures and all check to make sure that the HPLC method used follow good chromatography fundamentals (e.g. Proper K prime, peak shape, sample is dissolved 100% in mobile phase before injection, column washing with a solvent that is stronger than the mobile phase after each analysis, etc). If your samples have chromophores present, then your HPLC system should have a DAD inline with the MS detector. The combination of detectors is used to collect the most orthogonal data about each sample (UV/VIS, retention times + MS fragmentation data) AND can be very useful to troubleshoot problems.

Speak with a local professional chromatographer, on-site, to find a solution. The LC-MS system is designed to be used by someone with many years of professional training.

Here is a publication on this topic Ekta:

Lawlor K, Nazarian A, Lacomis L, Tempst P, Villanueva J. Pathway-based biomarker search by high-throughput proteomics profiling of secretomes. J Proteome Res. 2009 Mar;8(3):1489-503. doi: 10.1021/pr8008572. PMID: 19199430.

The peak rating is an important quality control measure that enhances the reliability and robustness of metabolomics data analysis by filtering out low-quality peaks and focusing the study on the most trustworthy metabolite signals.

Thank you so much for giving me a clear answer.

Phil Käding thank you so much for your help

Hi,

And welcome to the wonderful world of chemistry and LC-MS!

Please be aware that preparing your mobile phases is probably the easiest step in the process of acquiring LC-MS data, so be prepared for more, and more complicated, challenges..

What flow rate will you be using in your LC method? Together with the length (time) of the overall method, including gradient, column washing and re-equilibration, and the number of blanks, controls and samples you're planning to run, you can estimate the volume of both mobile phases you'll need. Always make a bit more than you absolutely need, as it will be impossible to completely empty a glass bottle.

With regard to preparing the mobile phases, for 1 liter, prepare a stock solution of 1 M ammonium formate in water by weighing and dissolving 630.5 mg ammonium formate in 10 mL water.

Then, for mobile phase A, add 1 mL of the 1 M stock solution to 998 mL water, add 1 mL formic acid and mix. Done.

For mobile phase B, I assume you also want 0.1 % formic acid, instead of 1 mM?

Do the same: add 1 mL of the 1 M stock solution to 998 mL acetonitrile, add 1 mL of formic acid and mix and you're ready to go. Mobile phase B will also contain 0.1 % water, but that will not be a problem (I guess).

Hope this helps: good luck!

Send email request on along with details of nature of samples, number , what type of study etc......

dr.daveusha@gmail.com

My mobile- +91 9820693161,

As mentioned above by Dr. Albert Lee, you can use protein A or protein G to remove heavy chain. You can also use cation exchange chromatography to separated light chain from heavy chain, especially when the amount of the light chains you need is small since you can use an high resolution analytical cation exchange chromatography column.

Thank you for all this valuable information.

In response to Devin Camenares , from the end of my T7 promoter to the first start codon, there are only 68bp. I've checked as you suggested, but no other start codon is localized in that region. Although I didn't obtain the sequence from the LC-MS results, the sample underwent a two-step purification process (Ni-NTA followed by SPFF), and verification was performed via SDS-page. Therefore, I believe it's my protein and not a contaminant.

For some supplementary information, my plasmid was ordered. I've checked the frame and sequence, and it seems correct. I will test the protein production with another strain of BL21 to see if the problem could arise from my strain background

In general, the best LC/MS system for analyzing surfactants will depend on your specific analytical requirements and preferences, so it is essential to evaluate different systems based on factors such as performance, ease of use, sample compatibility and workflow. In addition, consulting with experts in the field and looking at user reviews can provide valuable insights into the strengths and limitations of different systems.

Hey there Abdulwasiu Salaudeen Olawale!

MetaboAnalyst 6.0, huh? That's a solid choice for your untargeted metabolomics studies on soil and plants. I've used it before, so I can definitely give you Abdulwasiu Salaudeen Olawale a hand.

First off, if you're looking for a tutorial video, I suggest checking out the official MetaboAnalyst YouTube channel. They've got some great resources that can walk you Abdulwasiu Salaudeen Olawale through the process step by step. Also, the MetaboAnalyst website itself has detailed documentation that's worth a read.

As for running it on your Abdulwasiu Salaudeen Olawale LCMS/GCMS data, let's break it down:

1. **Data Preparation**: Make sure your Abdulwasiu Salaudeen Olawale data is in the right format. MetaboAnalyst usually prefers data in common formats like CSV, Excel, or TXT.

2. **Upload Your Data**: Head over to the MetaboAnalyst website, sign in, and upload your Abdulwasiu Salaudeen Olawale data. Follow the prompts to select the appropriate analysis type and upload your LCMS/GCMS data.

3. **Parameter Settings**: Set your Abdulwasiu Salaudeen Olawale parameters according to your study design and the type of analysis you're doing. This includes things like normalization, scaling, and statistical tests.

4. **Analysis and Interpretation**: Once your data is uploaded and parameters set, run the analysis. MetaboAnalyst will crunch the numbers and generate results for you Abdulwasiu Salaudeen Olawale. Take some time to interpret the results and see what insights you can gather.

5. **Collaboration and Publishing**: Absolutely, collaboration sounds like a great idea! Once we've got some solid findings, we can definitely work on publishing together. It's all about spreading knowledge and advancing science, right?

Feel free to reach out if you Abdulwasiu Salaudeen Olawale need any specific help with any step of the process. I'm here to make sure your metabolomics study goes smoothly!

Certainly! Here are some software options for analyzing LC/MS metabolites efficiently:

1. XCMS

2. MZmine

3. MetaboAnalyst

4. Progenesis QI

5. MassHunter

It seems like you're experiencing fluctuations in mass accuracy for the 520.9 m/z precursor ion during negative tuning on your Sciex X500r qToF. While this may not directly impact peak intensities or width, it could affect data reliability. Here are some steps you could take to address the issue:

1. Check instrument conditions.

2. Verify calibration.

3. Review tuning parameters.

4. Ensure proper sample preparation.

5. Consider external factors.

6. Consult Sciex support if needed.

XCMS online remains the standard tool.

Since you have them already frozen in liquid nitrogen you can probably skip the lyophilization step. Grind them in a ball mill to get a nice powder. As Kai points out, you'll probably need water in your extraction and definitely need it for your analysis if you use HPLC.

Hi Keller, Thanks for your reply!!

If the extinction coefficient is the same at the same wavelength, you might be able to use the same standard for two different compounds. Otherwise, you need separate calibration curves. If the compounds have different extinction coefficients, they will have different concentration/absorbance response curves (which should be linear). If different wavelengths are used, the lamp and detector combination will cause different response to a compound, and the extinction coefficient will likely be different. On for the mass spectrometer, different compounds may well ionize or fragment differently, causing differing response from the MS. To be sure that the two compounds behave sufficiently similar to one another, one needs to run the experiment to prove the correspondance.

0.001 ppb, and 0.0001 ppb..these are not reachable concentrations for MS systems even if you apply enrichment steps using large-volume SPE I guess...

Since the question is very not clear to me I should indicate that You need to develop a method by proving the working limits of the extraction are in alignment with the range of detection concentrations. Meaning, that extraction efficiency between 10 to 50 ppm is not something that validates 0.1 ppb, 0.001 ppb, and 0.0001 ppb levels.

Okay, no problem.

Hello, a bit more information would be useful to help diagnose the problem but just looking at the fronting peak shape my guess would be a sample solvent that has an eluotropic strength that is too high combined with an injection volume that is also too high. Try injecting a lower volume or matching the injection solvent with your starting gradient conditions.

Dear Andriy,

First, congratulation with your future instrument. I personally love Agilent 6546, I did not work with Revident though.

Second, in my opinion, the most important step in untargeted metabolomics by HRMS is to fully understand what it can and can't do. Basically, I think a researcher has to accept that untargeted metabolomcis will not provide meaningful results immediately. Also, one has to accept that a whole process is so complex (not difficult, but complex), that it will be sub-optimal for most of the chemicals of interest. Therefore, for example, I would not consider APCI as a must have source. In contrast, for the targeted analysis, APCI can be essential in some cases.

The software provided are kind of repeat each other (in my impression), so I ended up using mostly MPP for all tasks. I think it is quite nice, intuitive and powerful. I do prefer to perform post-processing data analysis using external soft, but build-in functions work well for the initial results.

I do recommend to have PCDL, it can save you a lot of time for the primary metabolites annotation on the fly. But you can use external soft and databases too, if PCDL is beyond your budget or so.

Maintenance of 6546 is acceptable. in my case I have to prepare tuning mix by combining purchased components, but I heard Agilent is going to (already did) provide prepared mixes. Cone cleaning is easy, the needle adjustment can be tricky, but hopefully you will not need it often. I also like Agilent's video instructions., but I strongly recommend you to make sure how good is Agilent support in your location. Problems are unavoidable, I had a great support in this cases, but it is in the US. I know that other locations can be different.

Good luck.

Hello Talha Baig

it would be desirable if you could show the presumed structure of the target molecule here. The measurements are ESI positive measurements, that take from the spectra, the mass 515 can be the target mass if the molecular weight is 514. With ESI positive mode an M+H adduct is formed, so you get a mass os 515.

Noel W Davies has noted that the isotopic pattern of masses in the 775 and 910 range shows multiple halogen atoms, the pattern at 775 looks like a Cl5 pattern to me, the pattern at 910 looks like a Cl6 pattern. Since the isotope pattern at 515 shows no halogens, the question arises where the halogen compounds come from?

As these are very small signals, they could also be impurities, but the suspected target signal at 515 is not particularly intense either

With a target structure and the reactants that were used, many things could certainly be clarified better, but try the approach of Hediye Erdjument-Bromage and calculate the exact mass of your target molecule and then try to find the signal in your measurements. Don't forget that M+H or M-H adducts are formed, alternatively M+Na (= M+23) for ESI positive.

Good luck and all the best

Joachim Horst

Somewhat less complicated than LC_MS I would suggest reversed-phase LC in combination with UV detection at 218 nm employing a proper (thus not to steep!) acetonitril gradient using 0,1% TFA.

No. "MS" data is only as good as the overall method used to collect and evaluate it (The "method" includes the column, mobile phase, instrument settings, standards and proof that the method follows good chromatography fundamentals). With no standards for comparison and other orthogonal techniques used, no identification would be possible. Purely speculative.

MS data on its own has very little value as a MS system will always output "data". Data "from other studies" would be anecdotal at best as only data collected on the same instrument, by a trained operator (which takes many, many years) using standards with an approved method would be useful to draw conclusions from.

Chau Phuong Bui: You did not provide any information regarding the make, model and type of instrument or autosampler in your post. When new users such as yourself post general questions such as this one, please take the time to first include such basic information in your post. There are several hundred different types of autoinjectors and samplers on the market and many operate and function differently. Also, please share with us the specific instrument errors recorded in the instrument software for the failure noted.

That said, there are many reasons why an Autoinjector NEEDLE may be bent during operation.

Here are a few reasons why the needle may be bent at 90 degrees on an AutoInjector:

Please contact someone with professional experience in maintaining and servicing the specific instrument you have to assist you in troubleshooting AND/OR contact the instrument vendor's service dept.

You can try a micro Kjeldahl and capture all the Nitrogen atoms as ammonia gas.

Batoul Alallam The reference to the paper seems to have escaped:

Plant Cell. 2011 Jul; 23(7): 2477–2482.

doi: 10.1105/tpc.111.086272

PMCID: PMC3226225

PMID: 21771932

I confirm that the links to supplementary material don't work.

In my opinion, this paper doesn't entirely meet the requirements of the metabolomics community. In particular, the term "annotation" should surely indicate that when a metabolite - identified or not - is found again in a later study, there can be no doubt at all that the same compound has been detected.

By that definition, as proposed in particular for steroids by Shackleton's group (2010), it is rarely possible to annotate a metabolite using LC-MS, however elaborate the mass spectrometer may be.

I have discussed this question in two diatribes published on RG about the failure, during half a century, to identify a putative endogenous inhibitor of Na,K-ATPase:

Putative inhibitors of the sodium-potassium pump in mammals: a review. April 2018, DOI: 10.13140/RG.2.2.10992.35845

'Endogenous ouabain (EO)' in mammals: absence of valid experimental evidence. December 2019, DOI: 10.13140/RG.2.2.10144.53765

I cited an earlier article, which may be useful here:

Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, et al. Proposed minimum reporting standards for chemical analysis. Metabolomics 2007 Sep 19 ;3(3):211–21. DOI: 10.1007/s11306-007-0082-2

However, I consider that this important subject, which is as old as chemistry, ought to be a matter for learned societies, particularly our chemical societies.

You need to fully understand the property of your target analytes and your marrix interference in your sample that you want to keep or get rid of. In your case, your targets are low concentration of the combination of neutral, acid, and basic small molecules. Your matrix interference are high concentration of large molecules of protein/peptides/phospholpids in the serum. You have at least two options to eliminate the majority of proteins by using a) acetonitrile crash (mix 1 part of serum and 3 parts of acetonitrile. Protein molecules will precipitate out of the solution and your analyes will be in a solution of 1:3 water:acetonitrile. The second way is SPE from Phenomenex or Waters or Agilent that will capture protein/phospholipids. Please do the Google search on these products. . Once you get rid of protein/ phospholipids in the samples, you need to capture your analytes and eliminate small polar/ionic compounds to minimi the matrix suppression at the Ion-spray interface of your LCMS instrument. The mixed mode SPE (MCX or MAX) will be a good choice because one cartridge will be needed. Please study further from the Waters application note of this product on the internet. Essentially if you use MCX, it has affinity for neutral/acidic compounds via reversed-phase mode and retain basic analyte via the cation-exchange mode. You diute the sample with warer to lowere the concentration of organic solvent in the sample and lower the pH with acid. At this condition, the acid analyte will not ionize and retain on the stationary phase along with the neutral ones. The basic analytes will ionizeband has positive charge and stay at the stationary phase. The small polar neut molecule such as sugar and small metal ions will pass thru. you can wash the SPE with water and may be 10% methanol/ water. To elute all anayte, you will use methanol having high pH (1% ammonia).

hope this helps.

Yes, it is better to know if the compound is volatile and thermally stable before you can run direct GC-MS . Also, run UV spectra from 200-400 nm to check if it has any absorbance in the range. If non-active you can only use HPLC with an ELSD detector to determine the purity first. There are other ways to find the structure such as NMR.

Great!

I figured the msms will give me high certainty as it being referred to as high degree of annotation.

Yes there was the original peak in this sample and dipeptides has a lot of shared fragments, which i did not realized before.

So Its just not the correct annotation. However im convinced its a dipeptide very close to Phe-Glu so I will get other isomers standard for that.

Thank you very much Jan

Murat Eravci Thank you!

Thanks a lot to all

Procedure: (I work in natural product metabolomics but not in biology. Therefore, it's crucial to be mindful of sample denaturation and proceed with necessary precautions.)

1. Slide Deparaffinization:

Deparaffinize FFPE slices by incubating them in xylene. Repeat this step until the paraffin is completely removed.

2. Rehydration:

Gradually rehydrate the tissue slices through a series of ethanol washes (e.g., 100%, 95%, 70% ethanol).

3. Proteinase K Digestion:

Prepare a proteinase K digestion buffer and incubate the rehydrated tissue slices in this buffer containing proteinase K.

Incubate the samples overnight at a suitable temperature to allow digestion of proteins and reversal of cross-links.

4. Nucleic Acid Extraction:

Extract nucleic acids using a phenol:chloroform:isoamyl alcohol solution. Perform centrifugation to separate the aqueous phase containing nucleic acids.

Transfer the aqueous phase to a new tube and repeat the extraction with chloroform.

Precipitate nucleic acids by adding isopropanol and washing with ethanol.

5. Washing and Resuspension:

Wash the nucleic acid pellet with 70% ethanol to remove impurities.

Air-dry or speed-vac the pellet and resuspend it in RNase-free water.

do you have LC-MS/MS data, or run standards.

To better understand what you are asking, please first familiar yourself with the basics of HPLC and also HPLC-MS (LC-MS) analysis. Please contact a professional LC-MS laboratory or experienced industrial chromatographer at your school for assistance. No one can provide instruction in "how to perform LC-MS" experience via a 'web forum', a class or even after a years worth of training. Using an LC-MS system is not the same as learning how to use a spectrophotometer. Learning just the basics of this complex technique just for one group of compounds and/or one mode of chromatography can take many years of full-time work.

As a student, your time may be best spent learning how to work with someone who already has this experience and can help you directly with your project. This will increase the chances that the data YOU collect and any interpretations made are accurate.

The above process will also save you the most time and teach you an important skill (**how to work with other scientists who have the years of experience in areas outside of your own).

Contact me

It is better to clean the MS system by using Alconox...mild and MS spec compatible detergent is efficient in removing aggressive contaminants such as sticky and hydrophobic moieties...

Disassemble all configurations which have a role in ion flux including the quadrupole if possible (it is applicable for Exploris and Qexactive). Follow the maintenance guide to avoid any failure...

For LC it is better to replace the tubings, valves, injection needle, and loop but since you figured out this is not related to the autosampler only replacing the tubings with new ones may be applicable.

Instead of zero injection or preview run just collect the data using infusion mode to see if there is any TAG signal at your MS spectrum. This will clarify the residue that exists in MS or LC...

Besides you may inject lipase at the desired pH and buffer composition several times to convert TAGs to more cleanable counterparts (e.g. glycerol and fatty acids) if the interference is available at your LC configuration...

Best...

For this you can see some available protein databases and tandem -LCMS data from various mass spectrometry sites.

Hi. This book, page 179. check it https://investigacionfitopatologiaumar.files.wordpress.com/2016/06/plant-propagation.pdf

There are several possible reasons why no emulsion formation may occur in a beta carotene/linoleic acid bleaching assay. Some potential reasons include:

1. Inadequate mixing: If the beta carotene and linoleic acid are not mixed thoroughly, emulsion formation may not occur. Proper mixing is essential for the formation of an emulsion.

2. Incorrect ratio of beta carotene to linoleic acid: The ratio of beta carotene to linoleic acid may not be optimal for emulsion formation. Adjusting the ratio of these components may be necessary to promote emulsion formation.

3. Insufficient energy input: Emulsification often requires the input of energy, such as through stirring or homogenization. If insufficient energy is applied, emulsion formation may not occur.

4. Incompatible solvents: The solvents used for dissolving beta carotene and linoleic acid may be incompatible, leading to a lack of emulsion formation. Using compatible solvents or adjusting the solvent system may be necessary.

5. Contaminants or impurities: Contaminants or impurities in the beta carotene or linoleic acid samples could interfere with emulsion formation. Purifying the samples or using higher quality reagents may be necessary.

6. Temperature and pH conditions: The temperature and pH conditions of the assay may not be suitable for promoting emulsion formation. Adjusting these parameters could help facilitate emulsification.

7. Other factors: Other factors such as the presence of surfactants, stabilizers, or inhibitors could also affect emulsion formation in the assay.

It is important to carefully troubleshoot and optimize experimental conditions to promote emulsion formation in a beta carotene/linoleic acid bleaching assay.

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ChatGPT DALL-E Bot, [11/13/2023 6:58 PM]

Selecting the solvent for extracting unknown compounds using a Soxhlet extractor can be a trial-and-error process. It is important to consider the polarity of the compounds you are trying to extract and choose a solvent that will effectively dissolve them. Additionally, the boiling point of the solvent should be lower than that of the compound being extracted in order to facilitate efficient extraction.

One way to determine the appropriate solvent is to perform a preliminary thin-layer chromatography (TLC) analysis of the unknown compound. TLC can help identify the polarity of the compound and provide insight into which solvent may be most effective for extraction. By comparing the results of TLC with different solvents, you can determine which one yields the best separation and select it for use in the Soxhlet extractor.

It is important to note that while TLC can provide valuable information about solvent selection, it may not always accurately predict how well a particular solvent will perform in a Soxhlet extraction. Therefore, it may be necessary to conduct further experimentation with different solvents in order to find the most suitable one for extracting unknown compounds using a Soxhlet extractor.

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ChatGPT DALL-E Bot, [11/13/2023 6:59 PM]

To perform sunglint correction and water column correction on WorldView-3 imagery in ArcGIS Pro, you can use the following steps:

1. Sunglint Correction:

- To correct for sunglint, which is the reflection of sunlight off the water's surface, you can use the Atmospheric Correction tool in ArcGIS Pro.

- First, open the WorldView-3 imagery in ArcGIS Pro and navigate to the Image Analysis tab.

- Click on the Atmospheric Correction tool to open it.

- In the Atmospheric Correction tool, select the WorldView-3 imagery as the input and choose the appropriate parameters for sunglint correction, such as selecting "Sunglint" as a correction option.

- Run the tool to apply sunglint correction to the imagery.

2. Water Column Correction:

- To correct for water column effects, which can cause distortion in underwater features, you can use remote sensing techniques such as band ratio or spectral indices to account for water column effects.

- In ArcGIS Pro, you can use tools such as Band Arithmetic or Raster Calculator to create band ratios or spectral indices that account for water column effects.

- Once you have created the appropriate band ratios or spectral indices, you can apply them to the WorldView-3 imagery to perform water column correction.

By following these steps, you can perform sunglint correction and water column correction on WorldView-3 imagery in ArcGIS Pro to improve its accuracy and usability for various applications such as environmental monitoring, coastal management, and marine research.

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ChatGPT DALL-E Bot, [11/13/2023 7:03 PM]

Yes, topographic correction is needed for Landsat 8 level 2 products in calculating NDVI for mountain areas. Topographic correction helps to account for the effects of terrain on the reflectance values captured by the satellite sensor, particularly in areas with varying slopes and elevations. This correction is important for accurately calculating NDVI in mountainous regions, as it helps to minimize the influence of topographic effects on the vegetation index values.

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ChatGPT DALL-E Bot, [11/13/2023 7:07 PM]

To extract a sample for LC-MS (liquid chromatography-mass spectrometry) analysis, follow these general steps:

1. Sample Preparation: Begin by preparing your sample according to the specific requirements of your analysis. This may involve homogenizing or diluting the sample, depending on its nature.

2. Extraction: Choose an appropriate extraction method based on the properties of your sample. Common extraction methods include liquid-liquid extraction, solid-phase extraction, and protein precipitation.

3. Centrifugation: If necessary, centrifuge the extracted sample to separate the supernatant from any solid particles or debris.

4. Filtration: Use a syringe filter or centrifugal filter to remove any particulates or impurities from the extracted sample.

5. Transfer: Transfer the filtered sample into a vial suitable for LC-MS analysis.

6. Storage: Store the prepared sample in appropriate conditions (e.g., refrigerated or frozen) until it is ready for analysis.

It's important to note that these steps are general guidelines and may need to be adjusted based on the specific requirements of your LC-MS analysis and the nature of your sample. Always refer to the standard operating procedures and guidelines provided by your laboratory or analytical instrument manufacturer for best results.

Two things spring to mind. One is that the protein has a natural proteolysis site near the C-terminus which is resulting in most of the protein being processed to what you see. The second is that the actual clone you are using for expression has picked up a mutation so that protein translation is terminated or frame-shifted near the C-terminus. You can check the latter by either sequencing plasmid from the exact clone you are using for expression, or checking that an independent clone/transformant shows the same behavior.

Hi,

Although I'm not sure what the compound is you're detecting at m/z 241.2, it seems somewhat illogical to analyze phenol using LC-ESI-MS in positive ion mode, given the apparent absence of proton affinity in the molecule?

Have you tried using negative mode?

There is always a possibility, in a biological system, and presumably you do not know the stability or other characteristics of the molecule(s) in question.

Freeze drying, with the sample at reduced temperature, is the least likely drying method to cause degredation to your sample. Moving the dry sample to dry storage in a refrigerator, or better still, a freezer - the lower the temperature the better, is generally viewed as the safest.

The Thermo Scientific Q Exactive mass spectrometer is a popular instrument for proteomics research, but it is primarily associated with bottom-up proteomics, where proteins are digested into peptides before analysis. While it is possible to use the Q Exactive for top-down proteomics, it may not be as effective as other specialized instruments for this approach.

Top-down proteomics involves analyzing intact proteins without prior digestion into peptides. This approach can provide valuable information about protein isoforms, post-translational modifications, and protein complexes. However, it comes with its own set of challenges, as intact proteins are larger and more complex than peptides.

Here are some factors to consider regarding the effectiveness of the Q Exactive for top-down proteomics:

In summary, the Thermo Scientific Q Exactive can be used for top-down proteomics, and it offers high-resolution mass spectrometry capabilities that are beneficial for intact protein analysis. However, the field of top-down proteomics has advanced with the development of specialized instruments and methods, such as FT-ICR (Fourier-transform ion cyclotron resonance) and Orbitrap instruments. Researchers often choose these specialized platforms when focusing on top-down proteomics due to their improved performance and tailored features. Therefore, the choice of instrument for top-down proteomics depends on the specific research goals and the resources available to the researcher.

Periodic analysis of the sample can establish the stability of your sample. Most likely, your sample may have degraded.

Thank you Mr. Murat Eravci... This will surely help me out for the troubleshoots