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I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different treatment groups. I could either do an LCMS/MS or a Western blot. Which one would you recommend and would you do both for validation? I am looking for time- and cost-effective techniques. I'll appreciate any suggestions. Thanks!

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    $\begingroup$ How would you prep the tissue for LCMS/MS? That read out would totally look like garbage. There are all sorts of tricks for western blot that you could get an accurate measure. Personally, I would do ELISA to quantitate how much protein you actually have. $\endgroup$ – jwillis0720 Jun 22 '15 at 9:14
  • $\begingroup$ Which quantification do you need, absolute, relative, or semi? Usually semi-quantification would be enough. $\endgroup$ – 243 Jun 22 '15 at 12:40
  • $\begingroup$ Related post $\endgroup$ – WYSIWYG Jun 23 '15 at 4:08
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Western blot, though is a commonly used technique and is relatively simple to do, has some issues:

  • Low throughput: it is difficult to analyse multiple proteins simultaneously
  • Limited cross comparability: since antibodies to different proteins can have different affinities, they cannot be compared with each other.
  • Low sensitivity
  • Not very quantitative

LCMS addresses all the above limitations of western blot. It is also possible to do targeted proteomics i.e. study just some few proteins instead of the entire proteome. An LCMS system has a high initial setting up cost but a relatively lower run cost. However, the mass spectrometer has to be maintained and it is not an easy job; you basically need a facility and a dedicated technical staff.

Which one would you recommend and would you do both for validation?

Which technique to use depends on what you really want to see and what resources you have at hand. It is a commonly practised approach to do a western blot for re-validating a few selected genes identified from LCMS experiment, which is just to prove that the result can be obtained using another technique too.

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Although quantitative methods using MS have been developed, MS is not inherently quantitative. Quantification with MS could be quite tricky. Therefore, it is not the first choice. But, if you do not know which protein levels change and want to find proteins the expression levels of which are different between your samples you are going to compare, MS is not bad idea. In this case, validation is necessary. In other words, MS is not used to validate WB results in general.

In your case, you know which proteins you want to see. Therefore, I would suggest WB. This is a more direct way. Validation of WB may not be necessary, but it is better to get some supportive data: the protein activities in lysates, mRNA levels, etc.

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    $\begingroup$ MS=Mass Spectrometry; the name itself implies quantification. When you say it is not so quantitative, what are you comparing it with? $\endgroup$ – WYSIWYG Jun 22 '15 at 13:29
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    $\begingroup$ Peptides are ionized to determine the mass and ionization efficiency is not predictable. It would depend on peptide sequences and probably environments surrounding peptides. I think the word spectrometry in MS is used because you can see spectrum of molecular mass. $\endgroup$ – 243 Jun 22 '15 at 15:13
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    $\begingroup$ @VanceAlbaugh The only advantage of western blot is that it is easier to do and most people are comfortable with it. As you said, it is certainly cost-efficient and easy when only a few proteins are to be studied. However, even in this case cross comparisons are difficult. Antibody-protein interactions for different pairs are not comparable. It can be much more biased than fragmentation/ionization bias in MS. $\endgroup$ – WYSIWYG Jun 23 '15 at 4:12
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    $\begingroup$ What I am saying is that, despite all the possible problems you that mention, western blot is much more unreliable than MS. In MS ionization efficiency may vary but there are ways to detect multiply charged species (it also depends on ionization method and IMO only ESI has a high likelihood of producing multiply charged species). In western there is no way to compare two antibodies unless you make a standard curve (which I don't think anyone does). $\endgroup$ – WYSIWYG Jun 23 '15 at 5:44
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    $\begingroup$ Western blot is an old technique. MS has been relatively recently developed for proteomics so a lot of advancement is still going on. Stable isotope labelling helps in differentiating samples quantified in the same run (test vs treated). At least I know this is so for SILAC and ITRAQ. I said standard curve is necessary for WB because there is no way you can compare two different antibody labels from the same blot. This is turning into a huge discussion and perhaps we should discuss this in chat. $\endgroup$ – WYSIWYG Jun 23 '15 at 8:11
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The best way is to use FPLC if you know what kind of protein you're looking for.in case you don't know what are you looking for,then you can run a 2D-PAGE and after analyzing spots then use LC MS/MS to identify your proteins and then continue with FPLC ( for record FPLC is a method of HPLC or LC which is protein friendly and even you can use it to isolate and purify your chosen protein )

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