LC-MS - Science method

If you have a well-defined standard against which you can measure, it's a good method, if you have none yet, it isn't.

If you don't have any standard to compare with, the most accurate method is probably evaporating the solvent carefully and weighing what's left. If you have a mixture of species, you can also perform preparative LC and the continue with evaporating-weighing.

Sometimes, this might be happend. Following that, it could be interprated using a series of reasons and errores. Take a look to the following links.

Hi Dr.Haslina

I am interested in this topic please send me a message on my WhatsApp 00249911511696

Maybe this article helps: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8996309/ It mentioned the method of using molecular docking and molecular dynamics simulations to model, targeting ligands specific proteins.

You have a BUFFER solution with methanol ("10mM NH4Ac in MeOH,") so first you need to run a solution that is the same, but without the buffer present through the HPLC system to wash the buffer away (be sure to first check column compatibility with the solution, for each storage or just washing). Once ALL of the buffer has been flushed from the system to waste, you can begin to switch over to a new, fully miscible solution / mobile phase (e.g. ACN / Water). ***Please, before changing anything by yourself, ask your teacher or the person who has professional training in using the HPLC and/or LC-MS system for direct help.

Dear Najmin,

when you use detergent-based buffers for cell lysis you should make sure to include a clean up step prior LC-MS/MS analysis (e.g. FASP filter, C18 OMIX tips or in gel digestion), as non-ionic detergents may interfere with optimal peptide separation (as they tend to stick to your column). For tissue extraction optimal results can be obtained by freezing the tissue in liquid nitrogen and subsequently pulverizing the tissue using a pistil. The pulverized tissue may be solubilized in a detergent-based buffer as you tried before, or if you want to avoid any clean up steps with a urea-based lysis buffer.

Get rid of TFA or use mass spec grade TFA or better use DFA. Try to use only FA or AA for positive ESI not to see mentioned adducts. Ambic can be preferred as a negative ESI buffer that triggers deprotonation owing to its alkaline nature. It also produces multiply charged pseudomolecular ions...

Theoretically, the SIM method could obtain higher abundance peaks due to not losing the ions in the process of MRM transition. For instance, for higher m/z ions SIM could have high abundance with a relatively low noise signal. However, the MS/MS mode has advantages that should lead to MRM usage (noise reduction with the simultaneous confirmation of compound).

Sometimes the use of SIM is the only option. Some sterols-related compounds when using fragmentation give a very characteristic fragmentation pattern with no predominant ions.

BME works fine at similar concentrations to DDT. I don't use it anymore because BME gives me a migrane. You actually don't need to cap the resulting C's. As long as you keep the sample reduced (i.e., away from O2) you can use it directly in MS. I used to add a tiny amount of sulfite to the sample before ESI to preserve the reducing environment. However, as long as you can smell the BME you should be fine.

Things to consider: quantifying requires a quantification method, where you assign m/z, retention time, etc. to your analytes/compounds, so they got recognized by the software. In this case, you will get peak areas (or area ratio in case of using an internal standard), and this can be called qualitative analysis. For quantitative analysis, you need to add the concentration of each calibration curve standard in order to quantify.

You can follow LabSolution training or watch tutorial videos on how to quantify using LabSolutions software.

Thank you for your answer

Hi Noel W Davies,

Sorry for not responding until now (busy days).

Can you please explain how it well help?

To answer your question regarding "column bleed". Not if the functional groups on the silica are BONDED to the support, as most are... If the column support consists of material that is not covalently bounded, then it may indeed slowly elute off the support and out of the column with the mobile phase. There are HPLC columns with coated supports (very delicate and require special treatment to use). These "coated" supports will wash off over time and as the coating is washed off, their retention characteristics will change too. However, "coated" phases are not common. Most HPLC column types you are likely to encounter today have fully bonded supports. Any observed "bleed" is likely to be the result of poor method development resulting in material from the flow path, fouled column, sample(s) and/or mobile phase additives being seen at the detector. With LC-MS, adducts are very commonly observed.

*I should also mention that one rare thing that we do sometimes detect when using columns packed with very small particles (~ 2.1 u or less) is the silica itself. The LC column-end fitting has a frit (filter) on it to prevent the silica from leaching out into the flow path, but these extra small particles are often accompanied by super small "fines" which get past the filters and into the flow path (and detector). Silica and the associated chelated metals that they can attract are sometimes observed.

Mrs/Miss Enriqueta casal-banciella

There is not needed derivatization-step.

Please, concentrate on the content on the work (ref. [1]) cited in the following discussion, when it is appeared online available:

[https://www.researchgate.net/post/HPLC_RID-6A_and_UV_Vis_SPD-10AV_detector_Agilent_Hi-plex_H_column_RT1_12_3_min_RT2_65_min_Which_compounds_it_may_be].

Cryo-vials

The cryo-vials is manufactured for stability in extremely cold storage conditions

You can find what you are looking for, in OEKO-TEX standards.

AS ATTACHED

Hi William, thank you for this! I will have a look into changing the parts you mentioned. The wash bottles I was addressing were the ones that are linked to the multi wash system, but yes I also changed the seal wash solvent. I will additionally contact Agilent in response to your recommendation.

what kind of compound groups are you targeting to analyze? I assume it's a kind of hydrophobic matrix, therefore GC-MS profiling would be better. Indeed you may prefer LC-MS for a non-targeted approach. You need to narrow down the purpose first in terms of molecule polarity and size to find the best match...

N-linked glycosylation can be removed by treatment with PNGase F enzyme.

NEB also supplies a protein deglycosylation kit:

You may need to play around with the needle depth setting in the method, so the needle would go fairly close to the bottom without actually touching the bottom. Also reduce the draw rate so the liquid on the side wall has the opportunity to coalesce.

Selam Bora,

leave your peptides on the zip tips (after washing step without elution)and send them in an envelope at RT. The MS-facility can than elute the peptides from the C18 tips and start LC-MS analysis. I have done this several times with custom made C18 stage tips (much cheaper than ZipTips) in a collaboration with colleagues from Acibadem.

Let me know if you need further assistance.

Good Lick,

Murat

Hi,

the best way to select the polarity in LC-MS analysis is to try to inject your compound of interest in both polarities and observe which of the two ones gives the most intense signal and the highest S/N.

There are general rules to select the polarity:

NEGATIVE POLARITY --> molecules already charged negatively, molecules containing phosphates, acidic moieties, molecules with many Oxygen atoms, molecules contaning phenolic groups ecc.

POSITIVE POLARITY --> proteins, amines, molecules already charged positively, e.g. anthocyanins...

BUT the best way to select polarity still remains to try both polarities and select the most suitable to you.

BTW, if you are interest in a specific compound, look at the literature, there will be surely information about the experimental conditions, including polarity

Hi, Florian!

How are you?

I'm from Brazil and trying use PTAD to vitamin D derivatization. Do you have some news about your question?

Hi.

I see that many papers are analyzed using external standard methods.

Are you concerned about the recovery rate from the analyte?

I think you have a few compounds to choose from following articles.

baicalin

catechin and kaempferol.

quercetin

biochanin A

Anyway, of the polyphenols that would not be in your analyte, you can use those that are relatively close in nature and available in high purity. I believe you can solve this problem by selecting the appropriate one that is not contained in the analyte.

And I believe the topic someone else posted earlier may be helpful...

Good luck！

Most photosynthetic microbes can also fix nitrogen (e.g., cyanobacteria). Nitrogen fixation requires energy, which is generally obtained in the form of sugars from the roots of a plant. Therefore, the easiest way to screen is to use a nitrogen-free media (e.g., minimal media with no organic amines or ammonium salts) with sugars present to provide the energy. Growth means the cells must be able to fix N2 from the air. Of course, if the organism needs some other nutrient to grow, you won't know if it could fix N2 in symbiosis with a plant root.

Hi, u can check this link its will help

Thank you so much Vishma Pratap Sur and Rob Darrington for the suggestion.

Actually, reading the question with very strict meanings, he might be looking for a conversion to grams. If the density is known, multiply the volume by the density to get the weight. The concentration of a pure liquid compound is 100%.

Mobile phase pH, analyte purity, mobile phase combination can cause the peak splitting

You can contact ---- ICAR-Central Institute for Research on Cotton Technology, Adenwala Road, Matunga(East), Mumbai-400 019.  E-mail: director.circot@icar.gov.in

A few misc. comments :

The Metabolomics Innovation Centre (Canada) - www.metabolomicscentre.ca

Over 50 targeted assays (with absolute quantitation) and 6 global/ untargeted assays. Both NMR and MS based services are available, including assays specifically for plants studies. All prices/ assays are available online. Fell free to contact me directly.

Sample should be filtrated 0.45 or 0.2 um.

The rule is to inject in eluent or solvent that has lower or equal elution strenght that primary composition in the system.

regards,

GB

This paper may be useful:

Past 4 - the Past of the Future - Natural History Museum (uio.no)

You do not need software or have a "software" solution for your request of: "Now I need to identify compounds from the LC-MS chromatogram". The web will not provide you with answers.

LC-MS chromatograms alone are not reliable or useful to answer such questions posed or suggest next steps. The full method details, sample details, plus additional orthogonal data from other detectors would be needed for evaluation first, then the running of actual standards to make any qualitative ID's might be possible. This can not be done from the chromatograms. MS detection does not "detect" all compounds present and erroneous data can easily be generated when condition are not optimized and/or methods do not follow good chromatography practices (MS is NOT a universal detector and is completely dependent on the actual method used and the training of the instrument operator who developed it).

Please seek out some professional, local help with your project. A proper method of analysis can then be developed, optimized, data collected, standards run, data analyzed and conclusions reached.

a couple of articles that answer your question:

good luck!!!

Luca

Better to call an LCMS support engineer

Good luck.

Kindly visit...

Kindly check..

Kindly visit..

Vishnuvardhan Nomula check..

Good luck.

No (free or cheap) courses of which I aware. The best advice is to find a paper doing something similar to what you are doing that presents the data in a way that you find compelling. In terms of statistics, THE issue is convincing others at a glance that of the statistical range on the value(s) in question (i.e., variance or standard deviation about a mean). In terms of presentation quality plotting software, I recommend Kaliedagraph, which isn't all that expensive and has some good statistical graphic layouts. KG is limited to 2D, however. R is free, and does pretty well too, but has a steep learning curve to make presentation quality graphics. The nature of the data will dictate the presentation format you will use. Bar charts, box plots, ven diagrams, x-y-z linear, log-linear, or log-log, scatter plots (all with meaningful error bars), statistical plots. The big issues you face is that almost all the stats tools and graphing software is set up for Gaussian (parametric data). No one ever seems to check that their data is actually parametric,. however, which leads to lots of really bad conclusions. If your data is not parametric, you will need to look at non-parametric methods, and adapting the software to reflect that reality.

Kindly, Check the estimated yield of sample in the eluate of desalting column.

Kindly visit..

The standard for excluding a single data point is typically 95% (or 98%) confidence that it is beyond the standard deviation of the other cohort samples. The problem with most LC-MS/MS studies is that the standard deviationis so wide (because n is so low) that almost everything is within 95% confidence. PCA analysis is merely suggestive of what has the most variation between the cohorts. It is rarely statistically-valid at a given confidence level and always requires focused experimental follow up with more single biomarker-focused validation effort. When this is done, all the PCA targets typically disappear as aberrations. The basic issue is that biomarker concentrations cover a range of values for each cohort, these ranges typically overlap and you ultimately need to perform an ROC analysis to identify a useful biomarker, that requires 100's of patient samples.

Antibody generation for a single amino acid is a huge chanllenge indeed, how to obtain acceptable specificity and sensitivity for the generated antibody is a big issue. Here is also a paper that I was ever involved to the hapten design, where the the aim is to descriminate two similiar amino acids M from A, the difference for these two is alomost only half part of a amino. Fortunately, the hapten design strategy made important role, and proved to be ok finally. Maybe this paper could be referenced for your question. Good luck!

Suarez-Pantaleon, C; Huet, A. C.; Kavanagh, O.; Lei, H.; Dervilly-Pinel, G.; Le Bizec, B.; Situ, C. Delahaut, P.Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin. Analytica Chimica Acta, 2013. 761(1), 186-93

Please, cosider, also the following attachments, associated with reference [1], shown in my preceding posting:

(i) MTZ-urine-equ.tiff: It contains our innovative basic equations (1) and (2);

equation (3) is according to Arrhenius's theory;

and

(ii) MTZ-urine-dqc.tiff: It contains diffusion coefficients of MTZ in urine according to reference [1] and equations (2) and (3). As can be seen, there is excellent correlation between our theory and Arrenius's one.

Once, when there are assigned MS ions to corresponding 3D molecular structures, there is not needed employment in internal standard.

There is, in actual fact, excellent-to-exact liner correlation between two completely independent theories: Our stochastic dynamic formulas, mentioned above, and the Arrhenius's one.

Thus, 172.072 belongs to N³⁺-cation of MTZ, while MS peak at m/z 172.041 is assigned to N⁺O-cation. MS peak at m/z 171 belongs to cation-radical of MTZ.

The shown equations describe completely and absolutely mass spectrometric phenomena toward (a) quantitative and (b) 3D structural analyses of analytes.

In other words, even in complex matrix like urine, there is completely able not only to determine analyte quantitatively, but also to determine its 3D molecular structure; furthermore, exactly in chemometric terms.

Good luck.

I agree with Andre Fabris. ESI is a mild/soft ionization technique that performs ionization mainly by transferring or removing hydrogens. This typically ends as detectable hydrogen adducts of the molecule at positive mode as precursor ion. Besides other adducts, this formation needs proton acceptor-bearing molecules such as peptides. Basic amino acids such as lysine arginine histidine and other nitrogen-carrying side chains of peptides make it more prone to accept hydrogens thereby ionization at positive polarization.

Take a look at this for fundamental,

Dear Dr Naga Veera Yerra,

These softwares may actually help you (MetFrag and XCMS).

Here's their respective links:

Best wishes,

Sabri

As far as I know the "Type" feature only describes the spectrum type. E.g. 'MULTI-MATCH' = MS1 labeling cluster identified by matching between runs. [from: http://www.coxdocs.org/doku.php?id=maxquant:table:evidencetable]

So I assume your 13 RAW files were obtained from multiple measurements of the same sample. Or at least it seams that way if you only obtained a single evidence table. I think MQ returns the MULTI-MATCH type if you use "match between runs" for your search. Either way - I would say these peptides are valid as they were matched between multiple runs.

Are you sure these peptides don't have a precursor ion? What does "Charge", "Retention length" or "Retention time" say for peptides of the type "MULTI-MATCH". If you have values there - you detected a precursor ion for sure.

Again I would highly recommend to a address such a detailed question to the MaxQuant builders.

Whereas It is possible

It depends. Much data, for example fisheries records, are published for a given purpose - for example to manage a fishery. Many years after that data was published, it may provide other researchers with other information - for example on understanding "shifting baselines". There are many very good pieces of research using historic datasets. My herbarium vouchers are a form of "data". They are lodged in public Herbaria across Australia. The Herbarium staff make the vouchers, and the associated environmental data, available to researchers around the world. They do not limit the number of times any particular voucher may be used to provide a datapoint in someone's research. Those of us who contribute these data rarely hear about their reuse unless we subscribe to platforms like Bionomia. Over a career collecting environmental data I have much that I may never explore fully. I use platforms like the Atlas of Living Australia's BioCollect platform to host my old datasets. It is possible that one day someone may use the hypersaline lake diatom and physico-chemical data from Australia to develop a "diatom metric" index of water quality for these understudied habitats... or for gnammas, or coastal lagoons...

IAs you know what analyses you plan to subject the dataset to, maybe you would be better served in clubbing a group of papers together that look at the different things you have extracted from the dataset, and then provide the entire group to one journal to publish as a "set".

But I would not like for you to have the opinion that data may only be used once then needs be discarded. What a waste of effort. Well conserved datasets, with excellent metadata relating to the methodologies and data collection, can be valuable into the future, in ways we have no current understandings about.

This will be helpful. Thank you Nayan Chandra Mohanto and Udaya Jayasundara

You can also use free online tools for multivariate statistics: MetaboAnalyst (https://www.metaboanalyst.ca/) or Biostatflow (http://biostatflow.org).

p.

1) Most mAb target more than just one protein.

2) Consider the non-specific binding problem. If you are seeing just high abundance house-keeping proteins, this is likely the culprit.

3) It is possible to add protein crosslinkers to the solution before IP. Note, the diluter the better. Then you can use a specific endoprotease to digest the precipitated protein and look for a high abundance crosslink. Random crosslinking will result is chemical noise in the MS. Jonathan Amster (U Ga) did some work in this area with cleavable crosslinkers so that you could sequence the resulting peptides to ID the proteins. You can use a mass defect tagged crosslinker to help identify the crosslinked peptide pairs, but you will likely have to synthesize that yourself.

The difference is ionization efficiency. Some ions 'fly' better than others in the same mode in MS. There is lots of speculation about why this is so, which occasionally breaks out in shouting matches (reaching the point of fist-a-cuffs) at ASMS and other MS conferences. Consequentliy, I will leave my speculations out of this response. However, just like some ions will only be seen in positive ion mode and others only in negative ion mode, the differences in relative ionization between molecules in a single mode can easily give you the result you see. If you really want to see if the molecule is upregulated or downregulated, you can spike the sample with a 13C or 18O-analog of the compound and deconvolve the isotopic patterns quantitatively. see reference attached.

Eef Dirksen Thank you. That's an interesting idea. I have two large data sets so it's impossible to do it for all but maybe for a few chosen ones. I will look into it.

Back washing of column through Acetonitrile, 0.1% phosphoric acid in water.

A photodiode array detector, or PDA, is a type of absorbance detector (such as a UV detector) that is able to detect light-absorbing compounds at the PG level. However, PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained result in two dimensions ( optical density and time), but PDA adds the third dimension (wavelength). The analyte or related impurities peak can be determined simultaneously during separation at all wavelengths, which is convenient for choosing the most suitable wavelength and determining the purity of the analyte or related impurities.

MS Mass Spectrometer

The analytes are detected based on their MW (Mass: charge). The obtained information is especially useful for compound structure and identification (based on the Mass: charge ratio (without standard injection)). By constructing a standard curve, a very low detection limit of analyte can be quantified.

Hi Gaurav,

Which ionization source do you use in LCMS?

In LCMS sytems, extra peaks are common and this doesnt always mean that you have a big impurity.

As the ionization efficieny of analytes greatly depends on their structureand the matrix, Seni quantitative approach generally doesnt work well.

There may be several possibilities:

-The impurity may ionize more efficiently than your analyte.

-Dimer, trimer formation may be occured.

To clarify, you can change the gradient and check if retention time of unexpected ion changea or not. Secondly, you can use isotope labelled standard to observe the possible adducts of your analyte.

Regards,

Check https://elucidatainc.github.io/ElMaven/faq/

In my humble opinion, it is better to HPLC and LC/MS. The GC/MS does not really favorable results for flavonoids and phenolic compounds. It is essential to prepare the respective standards solutions and draw the calibration curve before starting the test. It is strongly recommended that it was prepared some prototypes and the result obtained were compared with principal samples/analytes.

Dear Francis, apparently it helps reading you original question carefully. You can e.g. search the internet for terms like "LC/MS service in Nigeria". This provided for example the following address in Lagos:

I also suggest that you search the various departments of your own institution, the University of Port Harcourt, for researchers who perhaps have LC/MS facilities, e.g. the Department of Pharmaceutics and Pharmaceutical Technology. See e.g. the following potentially useful article:

(please see attached pdf file)

I hope this helps. Good luck with your research!

Check https://www.rsc.org/images/AMC%20LCMS%20Guide_tcm18-240030.pdf

MS is a technology that identify substances by their molecular mass. To do so, the substance is first ionized and then fragmented into small fragments by collision with an inert gas. The mass detector at the last chamber of the MS will detect the mass of each fragment that arrives at the detector.

This is an introduction to MS, from which we understand that for sure we need collision to happen in every MS (tandem or single).

I am not aware of an MS that detects only molecular ions (parent ions) and identify substances accordingly. Many substances share a similar Molecular mass which makes it not differentiable in the MS without another criterion for identification, which is why fragmentation is used to make unique fragments for the purpose of identification of different substances in one sample.

Who said there is no collision in single quad MS??

Thanks, Alexey! I got it now with a tiny drill. Silly thing though! Take care!

Hi Hayder,

The answer depends on what amino acids you are wanting to analyse. As a general method might not be applicable to all amino acids, if you have found methods that meet most of your needs but it is only the internal standard that you are stuck on then that is different.

I would suggest using a method you have found and using a generic internal standard. Labelled IS can be quite expensive but generally are much better as it goes through the same extraction.

Amino acids are generally polar and a couple of generic ISs that I would suggest are gemcitabine (cas number: 122111-03-9) and indomethacin (cas: 53-86-1). These may be a good starting point but they do come with some drawbacks. I would use a concentration around your mid QC level to start and adjust it from there.

You will need to make sure you perform a matrix effect experiment comparing your IS to all your analytes. This will allow you to check that the IS is suitable. A good IS will pass through an extraction in the same way as your analytes of interest. So this step is crucial to ensure that you dont have excessive variability and problems reproducing data.

I hope this helps

Check https://www.researchgate.net/publication/24191055_Determination_of_trehalose-6-phosphate_in_Arabidopsis_seedlings_by_successive_extractions_followed_by_anion_exchange_chromatography-mass_spectrometry

Dear Dr Duri Eun,

I highly recommend you to read this interesting book (in attached), since it summarizes all what you have to know, to improve your knowledge about Liquid chromatography-mass spectrometry, which will help you to perform this technique correctly.

Best wishes,

Sabri