Phosphoproteomics - Science topic

Perfect, thank you so much again for the help and the insights!

Hi Amanda,

if you've alkylated well, the vast majority of your Cys will be Cys-CAM, so treat that like plain boring Cys. ie. Cys-CAM is there and it's just another amino acid. You can check your level of alkylation by doing a search where Cys-CAM is a variable modification.

It's similar for MSO. That should be around the 5-10% of Met residues.

For this peptide:

EM(+15,99)LMEDVGS(+79,97)EEEQEEEDEAPFQEK

EMLMEDVGS(+79,97)EEEQEEEDEAPFQEK

Add the intensities to get one result.

Likewise if you see this version;

EMLM(+15,99)EDVGS(+79,97)EEEQEEEDEAPFQEK

Three versions of the peptide go into one result.

The hardest is this;

KRPQS(+79,97)PS(+79,97)PR

where you could also have

KRPQSPS(+79,97)PR

KRPQS(+79,97)PSPR

I'd treat the double phospho version as one result and combine the intensities for the two different monophospho versions.

I hope that helps.

We bought a column and popped it open to access the resin; Titansphere TiO2 (GL Sciences, Tokyo, Japan). I'm sure there are plenty of resins you can use.

Late answer but TiO2 and FeNTA are not suitable for phosphotyrosines( i.e I quantified 43 phosphotyrosines by a sample from which I quantified nearly 9000 S/T phosphosites so much less than 1% with TiO2) If you don't have sample quantity problems(not working on clinical data) pY100 antibodies are the best for phosphotyrosines. Not really an expert but if you can quantify more than 300 phosphotyrosines its a success.

Dear Fuat,

if you want to be sure, the only way to estimate the peptide amount is indeed a peptide assay. However, for most samples the amount of phosphopeptides is so low that overloading is rarely an issue, if your phosphopeptide enrichment worked good. Only if you amount of input material (based on protein assay) is well above 1mg it is worth considering to do the assay...

Good luck with your experiment,

Dominic

Hi Joshua,

the analysis does not have to be done with PD, it was recommended this was used. PD is an expensive commercial product, but is a very capable piece of software. MaxQuant is free and an excellent package fully capable of performing the analysis required in this instance. MaxQuant is also capable of working with PTMs, so yes, I'd start there. Good luck.

Thanks Mary Cristine Charlesworth , yes I do desalt with C18 columns prior to injection.

It seems everyone agrees the ultracentrifugation step can be substituted for conventional centrifugation.

This site is OK:

Bacterial lipopolysaccharide (LPS), a cell wall component characteristic of Gram-negative bacteria, is a representative pathogen-associated molecular pattern that allows mammalian cells to recognize bacterial invasion and trigger innate immune responses. The polysaccharide moiety of LPS primary plays protective roles for bacteria such as prevention from complement attacks or camouflage with common host carbohydrate residues. The lipid moiety, termed lipid A, is recognized by the Toll-like receptor 4 (TLR4)/MD-2 complex, which transduces signals for activation of host innate immunity. The basic structure of lipid A is a glucosamine disaccharide substituted by phosphate groups and acyl groups. Lipid A with six acyl groups (hexa-acylated form) has been indicated to be a strong stimulator of the TLR4/MD-2 complex. This type of lipid A is conserved among a wide variety of Gram-negative bacteria, and those bacteria are easily recognized by host cells for activation of defensive innate immune responses. Modifications of the lipid A structure to less-acylated forms have been observed in some bacterial species, and those forms are poor stimulators of the TLR4/MD-2 complex. Such modifications are thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. This hypothesis is supported by studies of Yersinia pestis LPS, which contains hexa-acylated lipid A when the bacterium grows at 27°C (the temperature of the vector flea), and shifts to contain less-acylated forms when grown at the human body temperature of 37°C. This alteration of lipid A forms following transmission of Y. pestis from fleas to humans contributes predominantly to the virulence of this bacterium over other virulence factors. A similar role for less-acylated lipid A forms has been indicated in some other bacterial species, such as Francisella tularensis, Helicobacter pylori, and Porphyromonas gingivalis, and further studies to explore this concept are expected.

You can try PathVisio? https://pathvisio.github.io/ easy to use and friedly interface :)

Merhaba Ayaz Anwar

You can also track the activity of a kinase through certain phosphorylations on other substrates, sites and proteins.

Or, a substrate can be phosphorylated by different kinases. Or its phosphorylation can be inhibited with different way.

There may be something during the preparation process that causes the loss of phospho sites. sampling and storage conditions may be effective. You can see the proteins (kinases and others) but the phosphorylations are gone.

I personally have always had great success with trizol. I use a similar protocol to the one found here:

Hi Katie,

You'll find in the links below an article that demonstrates the analysis of proteomics data using the Perseus software platform.

Hope this may help you.

Take an aliquote before drying the sample and try the "Dot blot method" using antibody against the most commonly occurring phosphopeptide. Since the same amount of protein is taken compare between before and after enrichment samples. Better is the blot in the enriched sample.

I have had success with this kit before illustrated in this paper.

SL-TMT: A Streamlined Protocol for Quantitative (Phospho)proteome Profiling using TMT-SPS-MS3

You need a lot of starting material, but I quantified around 2000-3000 phosphoproteins.

Dear Chandra Ramakrishnan ,

When it comes to your original question I think the interpretation of the prediction/calculation as you think yourself is: the most likely site is the indicated Ser (with apparently according to their database a slight possibility for the other AA).

Your related second question has to do with the term multiplicity.

In mathematics this means according to Mathwords: How many times a particular number is a zero for a given polynomial. For example, in the polynomial function f(x) = (x – 3)4(x – 5)(x – 8)2, the zero 3 has multiplicity 4, 5 has multiplicity 1, and 8 has multiplicity 2.

In Maxquant it means (something else?) as you can find here:

It states:

“Labels: The second tab is Group-specific parameters. You need to go there to specify your labels. The “Type” will usually be “Standard” (obviously), and the “Multiplicity” will be 1 for label-free quantification, 2 if you have light and heavy labels, and 3 if you have light, medium, and heavy. Any number of labels can be checked in the lists. In this example, the light sample is unlabelled and the heavy sample has been labelled with Arg10 and Lys8. (You can't see them both without scrolling.)”

PS. I am no expert of this program but I noticed an interesting discussion here on RG:

Further (background) information about the benefits and ‘problems’ of using these type of programs might be of interest as well:

Lee, D. C., Jones, A. R., & Hubbard, S. J. (2015). Computational phosphoproteomics: from identification to localization. Proteomics, 15(5-6), 950-963.

Good luck and best regards.

Hi,

I am currently working on such a tool. It will only visualize the different peptides contributing to a site and handles one user-specified phosphosite at a time. If that`s still helpful, I am happy to send you a beta-version.

I think you should lyophilize your samples thoroughly to remove the formic acid, then add water to return them to the original volume. Then add more trypsin because the original trypsin is probably inactive.

Yes samples are kept in wet ice.

The tubes for the bead beater also are kept in ice before and after you put the samples inside.

The plastic racks adapters are stored for 20 min in -20 freezer (i do it while I wait my tissues). That will keep the samples cool enough throughout the homogenization procedure.

I do not use lower temperatures because that freeze the tissue sample too much and it can't be homogenized well by the beads.

Hope this helps

https://academic.oup.com/nar/article/47/D1/D451/5150235

Although less elegant, you would also be able to do it excel or R or something similar. The main issue is that because you analyzed in one run, both base peptides and phosphor-enriched peptides will be annotated to the same proteins, and as such, you cannot distinguish directly. You could filter out all-zero proteins for one condition, but it depends on what you want to do with the data. If you really need to look at them separately, I would suggest running them again in two runs.

Your welcome!

Good Morning. They are activated T-cell blasts. (activated with anti-CD3 and anti-CD28).

You can also look here:

Pepsin is a random cleaver, so you end up diluting the signal by distributing it over too many random sequence lengths. Plus, you get different results with small changes in time and temperature. It's very problematic to work with. As pointed out in the above answer, mutliple digestions can improve sequence coverage. Chemotrypsin isn't the best since it has many cleavage sites. The most common pair is TRYP and AspN. Best results are to use individually and then in combination (three different digests).

Agree completely with Peter, the sample prep is crucial. If you don't get hat right it does not matter which LC-MS/MS you'll be using (the difference in performance between modern high res mass specs is not that big and the Q-exec you have access to will give you excellent results IF sample prep is good).

Have a look at these products for phospho-enrichment (http://www.resynbio.com/product_ti_imac.htm) as well as sample clean-up (http://www.resynbio.com/product_hilic.htm) and associated publications (http://www.resynbio.com/citations.htm).

Ran：As a young scientist, you may committed a common mistake by overly using too much amount of proteins. For any proteomics study, 100 micrograms of proteins are enough for analysis, along with proportional amount of trypsin or chymotrypsin. The solutions should be clear and no visible color shall be seen. Good luck.

Please "Recommend" if you find it useful :=)

Strait up decapitation and flash freezing.

Thanks so much for your answers. Those were so helpful.

Have a look at the MaConDa database. Note they use exact mass. I can’t see anything that would correspond to your contaminant, but double check the mass and scan the database to get an idea of what it might be. It at least should give you a place to start.

The Zhang paper has a spectrum with a prominent 524 ion (charge stage not specified) and it’s not mentioned in the text. Nevertheless, you should check it out.

X. K. Zhang, R. C. Dutky, and H. M. Fales. Rubber stoppers as sources of contaminants in electrospray analysis of peptides and proteins. Anal.Chem. 68 (18):3288-3289, 1996.

The Keller list is extensive (but no 524 ion).

B. O. Keller, J. Sui, A. B. Young, and R. M. Whittal. Interferences and contaminants encountered in modern mass spectrometry. Anal.Chim.Acta 627 (1):71-81, 2008.

perhaps the  uptake of the compound is inhibited in vitro by presence of something present in the cell culture medium, i.e. serum. Was serum present during your experiment? What is it the protocol?

you'll have to quantify separately the difference phosphopeptides. if you see 2 fold difference in one peptide and 4 fold difference in the other, you can say that you have 8 fold on that one site. however, you have to normalize it to the changes in the protein in the sample. it may be that the overall protein quantity has fallen and not the phosphorylation.

If you will properly select a concentration of glucose, you will avoid the risk of apoptosis. We worked with 30 mM of glucose using various cell types and there was no significant apoptosis. However, be careful because glucose may induce at this concentration accelerated cell senescence which will affect the phosphoproteome. 

Personally, I'd suggest you use the TPP via the Petunia interface (the web interface), as this is more user friendly than the CLI. If you have specific questions, try posting them on the dedicated Google group (see link).

HILIC could work but be careful when comparing phospho vs non-phospho due to 1) obviously ionization efficiencies of phospho are lower thne non-phospho so can not do direct comparison and 2) not all your non-phosho is present in the FG step from the TiO2 enrichment since some will be eluted only in the 2x ACN/TFA washes, so your non-phosho is likely to be under-represented

There are materials available commercially for depletion of albumin from serum-containing medium. Here are a few examples:

Hi Sulgi,

If you use RIPA buffer, as already pointed out, you will not be able to perform efficient in-solution digest and peptide clean up.  

However, if you have to use RIPA buffer, you can proceed to SDS-PAGE and therefore remove any detergents from the protein. After SDS-PAGE, you can then perform in-gel digest on the protein and extract the peptides for TiO2 enrichment.

Alternatively, you can solubilize the protein in 8M Urea, dilute the Urea to ~ 1M Urea and carry out in-solution digest with trypsin.  This workflow will result in peptides that can be purified and subjected to TiO2 enrichment.

Good luck,

Hediye.

Thank you very much, Vehary Skanyan!

Hi,

If I recall correctly, the best way to do this is by esterification using methanolic HCl. This is prepared in situ by the dropwise addition of acetyl chloride to dry methanol, thereby generating HCl and methyl acetate.

In this way, carboxylic acid side chains (and C-termini) are esterified, which improves the specificity of IMAC, that is used to enrich phosphopeptides. It can indeed also be used to differentially quantitate two samples.

Good luck!

Hi Jose,

though it's 3 years passed from the last post, was wondering if you could solve the problem? I have noticed when I make the gel, even before loading samples etc. when I look at the gel I see there is a smiling boundary in the middle of the  gel, it's like there is sth in phostag or manganese which make the acrylamide gel funny. Just was wondering if you solve the issue, and how?

thanks

Dear Dr. Oliván, ·

The following (two identical articles) papers describe a technique for assessing protein phosphorylation using nonspecific phosphatases that dephosphorylate many phosphoproteins in vitro.

 Shenolikar S. Detection of phosphorylation by enzymatic techniques.

Curr Protoc Protein Sci. 2001 May;Chapter 13:Unit13.5. doi:

10.1002/0471140864.ps1305s05. PubMed PMID: 18429117.

 Shenolikar S. Detection of phosphorylation by enzymatic techniques.

Curr Protoc Protein Sci. 2001 May;Chapter 13:Unit13.5. doi:

10.1002/0471140864.ps1305s05. PubMed PMID: 18429117.  

Best of luck!

Hi Lykke, 

If you are interested in predicting novel substrates that are phosphorylated by a kinase of your interest from a phosphoproteomics data, you can have a look at our recently developed GUI tool: https://github.com/PengyiYang/KSP-PUEL 

Cheers,

Pengyi

Dear Trung,

It looks like you do not have one database for the searches you performed on Mascot 5.  You have 16 databases, and they are all a bit different from each other.  How did you set your parameters? It is best if you opt for Swissprot and under species, choose Human for each of the searches.  If you save the parameters this way, each time you search Mascot, it should keep the same number of proteins/database to query your unknown dataset against.

The import into Scaffold requires THE SAME, EAXCT SWISSPROT database that was used for your Mascot search.  If not, you will have ? marks.  You can easily remedy this problem by going to "Experiment" on pulldown menu and "Apply New Database" activated.  You need to make sure the database you have used for the Mascot search is in your Scaffold Database folder.  If not, find Mascot SwissProt fasta file and under go back to Apply New Database, click on "Add Database", choose the Fasta file and Autoparse it while it is imported into the Scaffold Database.  When it is in the Scaffold Database, just Apply it and you should be able to get rid of the ? marks under Molecular weight of the Proteins.

Best,

Hediye

I would advise against eluting with 80% ACN as you end up with a lot of stuff that isn't peptides on your column. The maximum you should go is 50-60% ACN in our experience to have a cleaner peptide solution. 

OK, thank you so much. I will try.

Jose

Hey,

I agree with the above answers that genetic drift, the major concern with using cells of high number of passages is highly dependent on the cell type and cancer cells do have a tendency to mutate quite frequently. Of course once you start passing cells too long, things like mycoplasma, etc. also become an issue and it's best to start with a fresh stock.  But, for preliminary studies, that are backed up with tumor studies, it is acceptable to use cells with high passage number.

One exception however, if you culture cells directly obtained from primary tumor, you probably don't want to go beyond 2 or 3 passages at most. 

I've been in touch with the manufacturer's and this is what they had to say:

'The typical capacity depends mostly on the sample and the capacity of your LC-MS (since the elution can be directly ran on the MS). PolyMAC is unique in the sense that you do no need to adjust the ratio of PolyMAC to the peptide amount; the same procedure can enrich from fmol to mg sample amount. With this said, for your case, if the sample is hyper phosphorylated (e.g. stimulated), we don't suggest using more than 500ug per enrichment. 1-2mg mammalian proteins should be sufficient for other types of sample. If you need to enrich 3mg each time, you might try doubling or tripling the amount of PolyMAC and the capture beads used.

For your other question, PolyMAC itself is a soluble dendrimer which allows the reaction to be in homogeneous conditions (it's a clear solution). After the phosphopeptides are bound, PolyMAC and the bound peptides are then capture covalently onto the capture beads. This is a two-step process, but it only take 15min total time for both. Due to the first step being homogeneous, it offers numerous advantages for the enrichment (as described in more detail in the technical document and the publication). To use it, you just follows the protocol provided.'

So, I hought that it might be helpful for others to have this info, should they be interested in PolyMac.

Regards,

Prachi

We use 7M Urea/ 2M Thioures/ 2% N-octylglucoside, or If you have to use SDS or other kind of detergents you can follow the FASP (Mann's lab) protocol. In our hands works 10/10.

Hi Maria,

it very much depends on what kind of MS data you have and how it was generated.

Have you done any tandem MS of your peptides (CID, ETD, ECD)? This is what you need for identifying the position of your phosphorylation sites.

There's quite a few options in terms of automated data analysis, and selecting the right software is not always straightforward, particularly for phosphoprotemics due to the chances of misidentifying the phosphorylation sites.

Protein Prospector (http://prospector.ucsf.edu/prospector/mshome.htm) is an on line software you could try to search your data against a selected database (Uniprot, Swissprot and so on...). It will take data generated from pretty much any instrument (Q-ToF, low resolution ion trap, FT-ICR, with either ESI or MALDI as ionization source) as long as you can convert your raw data into an mgf file or equivalent to be uploaded on the software. This is something you should be abel to do by using your instrument-specific software for data analysis, which normally runs a script to generate the .mgf file (or equivalent).

Mascot is another very popular search engine which used MS data for peptides (and proteins) identification, although the free online version is limited to very small raw data files.

There are also more 'sophisticated' tools like Ascore which are particularly useful for distinguishing between isomeric phosphopeptides (it was originally designed for Thermodata).

Either way, a confident identification most of the times requires manual inspection of the raw tandem MS, as the search engine can sometime misidentify the right position of the modification. This is more than an issue with CID of phosphorylated peptides containing more than one S, T or Y residues which sometimes fails to generate enough sequence specific b and y ions for a confident identification.

I would also strongly recommend some background reading to have a feeling of what different search engines and different MS data can actually tell you.

As for the database, I normally refer to phosphosite plus http://www.phosphosite.org , which also includes whether a particular phosphosite was identified by MS.

Kind regards,

Francesco