Science topic

Proteomics - Science topic

The systematic study of the complete complement of proteins (PROTEOME) of organisms.
Questions related to Proteomics
  • asked a question related to Proteomics
Question
4 answers
Hi, I am proteomics resercher.
I ask for your understanding that writing in English may be lacking.
Because this is my first time to post question in the researchgate and i am not a native English speaker.
I am doing proteomics research comparing protein concentration(Label Free Quantification) in two groups using LC-MS.
We have 6 samples and two groups to analyze (3 samples/group).
we are planning to run only two samples in one day and other four samples several weeks later.
Is it possible to statistically analyze (ex, t-test) 6 samples together in this case?
I think it is impossible because Retention Time drift and machine performance change.
Please let me know your answer. (If you know manuscript about this issue, please let me know.)
Relevant answer
Answer
The issue you described is not a problem for proteomic analysis and your statistical tests will be valid provided you appropriately normalize the data. Most current proteomic search tools (e.g., MaxQuant, Fragpipe, ProteomeDiscoverer) will correct for mass and retention time drift. Of course, it is always best practice to ensure your instrument is calibrated before running your samples. Once you have protein-level data output from your search software, there are several statistical tools for controlling intensity (abundance) normalization. One of the more common methods is to perform Tukey's median polish. After you have performed some type of statistical test between your groups, remember to correct for multiple hypothesis testing using Benjamini-Hochberg or Bonferroni procedure. This will ensure you have adequately controlled your false-discovery rate (FDR).
  • asked a question related to Proteomics
Question
11 answers
I've conjugated a PEG4-maleimide (MW 613.66) onto an antibody fragment (52 kDa), but I'm trying to figure out how this affects A260/280 measurements on a Nanodrop afterwards. After conjugation and removal of unconjugated material on a PD-10 column, I measure the concentration of the eluted aliquots but the measurements seem really off, and the A260/A280 ratio is nowhere close to 0.5. I blanked the Nanodrop with the PD-10 elution buffer. Seems strange that such a small molecule could affect the readings that much. Does anyone have experience with PEG and if/how it could affect concentration measurements after conjugation?
Relevant answer
Answer
Cynthia,
Please contact me by email at
Sherman@mvpharm.com. I will try to "talk you through" the method of using the refractive index and UV absorption to calculate the protein and PEG concentrations without attempting a BCA assay or other chemical assay of the protein concentration. Our method is based on the Kunitani reference that I cited in this string of discussions in February 2015.
Best wishes,
Merry
  • asked a question related to Proteomics
Question
2 answers
I know this might be a bit too general question but:
In proteomic analysis (working with Perseus) when you deal with raw LFQ analysis. Do you always use Z- score? And do you always log2 transform your data?
It doesn't seem to me that is always needed. Besides, no matter if you do or don't the results on the graphs should come out the same, only differently scaled, right?
Maybe it's a dumb question but thank you regardless!
Anja
Relevant answer
Answer
Working with log-transformed intensity (or concentration) data is very convenient. Protein expression is approximately log-normal, so it's approximately normal at the log scale, where you can use the standard toolbox for analysis (i.e. linear models like t-tests, ANOVA, regression, ANCOVA etc).
I think visualization is also better done with log concentrations or log fold-changes. The resulting picture (and possibly the interpretation) should not depend on the perspective, that is, which group is chose as the "reference" and wich as the "experimental" condition. To give an example: you study the expression of protein X in males and in females. The fold-change male/female is 5 (wow what a drastic increase!), so the fold change female/male is 0.2 (eh, well, yes, some kind of reduction). The numbers look very differently impressive althought the express the very same difference (effect, ratio if you like). The (base-2-)logarithms are +2.3 and -2.3: same number, opposite sign. This is (imho) easier to understand and faithfully reflects the symmetry of the problem.
Using z-scores serves a different purpose. Here, z-scores are calculed from the log intensities, for the reasons explained above. The z-scores more closely resemble what a t-test or ANOVA "sees" from the data, as here the differences (from the mean) are related to the variability (the standard deviation). What gets lost is the information about the base intensity (expression level), what is often less interesting than the difference in expession levels between samples and groups.
  • asked a question related to Proteomics
Question
7 answers
Dear All,
I extracted protein from various tissues in mouse with RIPA buffer (added protease-/phosphatase- inhibitor cocktails and PMSF).
Quantified the protein concentration via Bradford and loaded equal total protein amounts.
My housekeeping gen is quite stable across my various organs but not 100% same.
For publication I would like to have a blot where all bands are equal - this is why I adjust the protein loaded according to the previous blot. With protein from cell culture this works fine but with a variety of mouse tissue organs I do not receive an equalized normalizer band.
Why is that? Is there anybody with experience doing multi-organ blots and has a good protocol or advise where to look for one ?
Thanks for any advise or help :)
Relevant answer
Answer
This could happen due to pipetting errors, or the proteins leaching out of the wells while applying on gels. The whole idea of using a house keeping gene is to nullify this effect. If you express the intensity of protein of interest with respect to that of the house keeping gene minor differences will not be a problem.
You could also try ponceau staining on your blots and can use that to normalize protein expression, if the house keeping gene does not work!
Do let me know if that help!
  • asked a question related to Proteomics
Question
2 answers
Which label-free proteomics method is best for quantitative proteomics analysis of extracellular vesicles: DDA vs DIA?
I have N=70 sample size of EV, Case A vs B.
Thanks.
Relevant answer
Answer
I beileve this is highly dependent on what you are seeking. DIA has better reproducibility and higher data completeness as Dr. Zepeda said, however, in terms of quantification, DIA has lower sensitivity than DDA as the complete spectrum must be scanned, reducing the acquisition time per data point.
I think DDA is suitable if you would like to perform A vs. B as it focuses on highly-abundant proteins.
  • asked a question related to Proteomics
Question
3 answers
Can you help with the issue of a better kit for the depletion of more abundant proteins (albumin, and all immunoglobulins) in the plasma of mice for use in proteomics analyses? Thank you very much.
Relevant answer
Answer
If you afford to use it, try the Multiple Affinity Removal System (MARS) from Agilent. Mouse Multiple Affinity Removal Columns and Cartridges are ready-to-use columns for simultaneous removal of major 3 interfering proteins Albumins, transferring, and IgG. Otherwise, you need to follow a relatively cheap, tandem and, long procedure as Dr.Wolfgang Schechinger indicated.
  • asked a question related to Proteomics
Question
5 answers
I have done SMD of protein at applying constant velocity using NAMD software and CHARMM ff. Since, this is my first time in performing Steered MD, I am not sure as to should I do umbrella sampling alongwith SMD? Are the results of SMD without any umbrella sampling significant ? It would be helpful if I could get some references as well.
Relevant answer
Answer
Sharmi Mazumder could you figure out how to pull and fix multiple atoms in smd ? i'm interested to know about the same.
  • asked a question related to Proteomics
Question
4 answers
I have tissue samples digested by SDS lysis buffer which i would like to use for lipidomics analysis. what do you suggest? do you believe is gonna be possible?
Relevant answer
Answer
You can't do this because the protein contamination has not been eliminated by the SDS buffer lysis, for this you will have to digest the sample with a protease enzyme.
  • asked a question related to Proteomics
Question
1 answer
Hi everyone,
I am trying to upload my proteomics data in Proteomexchange in "Complete Submission" type.
But the File validation (the first step after selecting data) is stuck in 50%.
I gave it time even for an hour, but nothing happened.
How can I solve the problem?
Thank you in advance
Relevant answer
Answer
Dear Mashid,
it is important to always use the newest version of the submission tool. Further an explanation of all steps is provided here: https://www.ebi.ac.uk/pride/markdownpage/pridesubmissiontool. If you are still experiencing problems PRIDE has a very good support team, which I am sure will help you. If you will not be able to manage the upload by yourself, they can even assist you in doing so. Their support/help email is: pride-support@ebi.ac.uk
Thumbs are pressed!
  • asked a question related to Proteomics
Question
10 answers
Is anyone aware of an SPE approach to isolating plasma metabolites, or at least substantially diminishing the protein/metabolite concentration ratio? The aim is to reach a balance of concentrations such that vibrational spectroscopic methods can pick up meaningful spectroscopic contributions from metabolites - contributions that are otherwise inaccessible as a result of overwhelming protein absorptions. Note that there need not be selectivity among the proteins - we are not interested in removing only the high-abundance ones (as is the case for proteomic MS work for example). Ideally, we want ALL of the proteins gone.
P.S. I am fully aware of the various lab methods in common use - crashing proteins out with organics (e.g. acetonitrile, methanol), and ultrafiltration methods. Interested in knowing more about less obvious options.
Relevant answer
Answer
Try to use Protein precipitation plates...It combines both protein crash and filtration which can be an automated and effective way of diminishing the protein composition.
  • asked a question related to Proteomics
Question
1 answer
I would like to ask you, different concentrations of glutamate (nitrogen source) have a great impact on the pH target of the Browning of fermentation broth. According to experience, whether to do transcriptome or proteomics
Relevant answer
Answer
I think this is depending on what is your aim of the research, since each one of them can provide you with specific information different from the other. So your aim will guide you to which one to do.
  • asked a question related to Proteomics
Question
4 answers
Could anyone recommend some good online Proteomics courses and/or books for both beginners and advanced students?
Relevant answer
Answer
Hello
please find the attached link
Principles of Proteomics
Best of Luck
  • asked a question related to Proteomics
Question
1 answer
I am using desthiobiotin instead of biotin for pulling down a particular protein via streptavidin beads. The desthiobiotin is clicked to the chemical probe I am using for the protein. Post enrichment, I am digesting the proteins 'on beads' and then eluting the desthiobiotinylated peptides. I am submitting the digested sample for proteomic analysis to determine site of modification. However, I am not sure if the desthiobiotin is intact during the proteomics mass spectrometry analysis and hence can't predict the exact mass difference expected.
Relevant answer
Answer
Hi Sauradip Chaudhuri,
If you do an on beads digestion, you will only get peptides which will be cutted away from the Streptavidin bead/desthiobiotin complex. Depending on the digestion enzyme and the available digestion sites on desthiobiotin you may also have some desthiobiotin peptides in your identified sequences after LC-MS analysis but this should not be a problem if you perform your LC-MS analysis and downstream data analysis in the correct way.
Good luck!
Murat
  • asked a question related to Proteomics
Question
1 answer
How can I reduce the viscosity of saliva samples for the proteomics project and homogenize the samples viscosely?
It should be noted that I do not want proteins to be removed in this way
Relevant answer
Answer
If it possible you can dilute the saliva with buffer to reduce the viscosity or you can use FASP for preparation the protein sample.
  • asked a question related to Proteomics
Question
3 answers
Long story short, I need to degrade 30ug of RNA and i need to do it at 4C. I want to use only as much RNAse A as necessary.
So if performing the reaction at 4C, how long of incubation time and how much RNAse A would i need to degrade 30ug of RNA?
For example, would 1ug of RNAse A(20ug/ml conc.) for 15min at 4C be enough?
Relevant answer
Answer
I agree with Kyle Skalenko that experimentation is needed but first contact the company to sort out whether they work in enzyme units or Kunitz units to define the activity of your rnaseA, Then assume that the activity drops by a factor of 2 for every 10c drop in temperature to get an approximation of how much enzyme or increased time will be needed
  • asked a question related to Proteomics
Question
9 answers
Hi,
I am looking for open source tools for pathway and network analysis for proteomics and genomics. Will appreciate tools with tutorials and simple to follow documentation.
Relevant answer
Answer
Hi Victor
begin with StringDB and DAVID, it's easy to handle.
all the best
fred
  • asked a question related to Proteomics
Question
2 answers
I have three MS spectra of an unknown protein. The protein has been separated and directly analyzed with a MS without trypsinization (top down approach). How can I know the identity of the protein? I suppose I can search for a matched spectrum in a library. However, I have no experience with top down proteomics and I don't know which software to use for the protein identification. Any help please?
Relevant answer
Answer
Hello! Thank you for your reply. I used MaxQuant but it is for shotgun approach. I was having a look to fragpipe but it seems it also can be used only for shotgun approach
  • asked a question related to Proteomics
Question
5 answers
For antibody sequencing
Relevant answer
Answer
It strictly depends on the overall approach to antibody sequencing. The main benefits of TIMSTOF instrument is high sensitivity and high ms/ms speed. It best fits to the transcriptome-assisted sequencing of native affinity purified pools( e.g. repertoire analysis of convalescent sera) It gives the acceptable quality ms2 of tryptic peptides with deep overall proteome coverage.
The same time timstof is inadequate instrument for true de-novo sequencing of mabs without database assistance. The modern Orbitraps performs multiple modes of fragmentation(CID, HCD, ETD, EThcD, UVPD) for different type proteolityc peptides. It greatly improves the quality of identification of long peptides (especially DMAPA-treated V8 peptides) and allows to differentiate Leu/Ile at the protein level. But this is true only for high-end Tribrid series Orbitraps(and partially for ETD hybrids) but not for Exactive and Exploris series.
  • asked a question related to Proteomics
Question
3 answers
What advantages does transcriptome have over proteome as the final product of gene expression is protein? Why to choose it?
Relevant answer
Answer
Not only it is much easier to work with transcriptomics but transcriptomics allow you to take into consideration the role played by non-coding sequences that are the majority of the genome and very often play a crucial role in biological regulation see for example:
  • asked a question related to Proteomics
Question
4 answers
This might be a trivial question and an operation do to, but I'm not experienced and haven't been able to find a direct solution online. I'm pretty sure I've overlooked something as this should be a simple task, so I'm asking here.
I have a list of some 1500 protein IDs identified in a proteomic experiment coming from a bacterial origin.
I would like to get GO annotation for those proteins so I could categorize them according to the "biological process"and "cellular function". Is there a web service or a simple program that could get those GO annotations ?
I'm confident that the GO enrichment analysis offered on the main page is not appropriate for the data I have and the information I want (I may be wrong), and my organism is not available in the list.
Does anyone have any suggestions ?
Relevant answer
Answer
Hi Dennis,
You may already found a workflow but just in case you not I would use STRING tool, you will need to enter a fasta file and select an organism and string will do the rest if your organism is not in their database just select the closest, it will give you a protein-protein interaction network and a lot of information (take a look to download section). Alternatively, you can use BLASTKOALA tool where you will also need to input a fasta file and you’ll get the GO annotations for your IDs.
hope it helps
Best
  • asked a question related to Proteomics
Question
13 answers
Dear scientists,
I got a set of around 4000 protein ids from a proteomic experiment and I would like to globally analyse if the particular groups of proteins in my experiment are significantly more hydrophobic and/or aggregation-prone compared with other groups. I am looking for an R programming library or a web tool that will enable me to obtain some quantitative value for hydrophobicity per protein for my sets. One thing I may do is to just simply calculate the sequence length adjusted number of hydrophobic amino acids C, L, V, I, M, F, W but this seems to be a little naive and I am not sure about the biological relevance of such a simple calculation not taking into account the whole structural aspects of the sequences...I would be glad for ideas on any smarter approaches...please help
Relevant answer
Answer
Basically, what you want to do is to determine the amino acid composition for each sequence (e.g. in this R package https://cran.r-project.org/web/packages/protr/vignettes/protr.html using extractAAC()) and multiply the number of times a given amino acid occurs with its hydrophobicity index in your chosen scale (see https://web.expasy.org/protscale/ for different scales) and sum up the values. However, in my experience, hydrophobicity is a poor predictor of the aggregation propensity of folded proteins, as aggregation is frequently linked to imperfect folding rather than to the association of properly folded molecules.
  • asked a question related to Proteomics
Question
3 answers
Hello everyone,
I'm planning on running my peptide samples on a high-resolution LC-MS instrument. I'm going to use a ZipTip C18 tip for the extraction of peptides and desalting of the sample. However, I'll be sending these samples overseas and it might take 2-3 days for them to reach their destination. I can potentially keep them in dry ice throughout their delivery, but it is very costly and we had few issues before where the dry ice evaporated until it reached the destination.
If I free-dry my peptide samples, do you think they are going to be stable for couple of days? Considering there won't be any humidity where the enzymes can work on the peptides, but I just wanted to get the opinion of people who has lots of proteomics experience.
Relevant answer
Answer
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
  • asked a question related to Proteomics
Question
8 answers
Multiplexed samples labelled with TMT tags. I am trying to quantify the ion intensity for each channel, however they are all being reported as 0.
Relevant answer
Answer
Hey Ben,
Don't know if you figured it out but we find we get better TMT results with FragPipe than MaxQuant (https://fragpipe.nesvilab.org). If it is a software problem give that a try. Normalizes across TMT plexs better.
  • asked a question related to Proteomics
Question
3 answers
Current search engines for MS/MS protein identifications such as: Mascot, MS Amanda, Sequest, etc., currently rely on the creation of a search library composed of computationally generated potential peptides through the cleavage by proteases (e.g., trypsin) of proteins from a given database. Different PTMs can be added to these computationally generated peptides, so that the search could be extended to address specific scientific questions, but this leads to significantly higher computational costs.
I have recently come across a case, where a highly enriched short protein could not be identified by a standard search, given that it was only generating a single peptide that had 2 fixed modifications. The modifications were not the most common there were and finding the right combination to use was time and computationally expensive.
I would like to open a discussion on the fact that pre-made peptidome libraries are a much better alternative to de-novo generated libraries of proteomes. Let’s get into the details!
As an example, I will use the ACE2 receptor, now infamously known to be the entry gate of Covid-19 into human cells.
The human ACE2 receptor undergoes a series of post translational event, such as: proteolytic cleavage by ADAM17 resulting in a soluble proteoform, glycosylation and phosphorylation of tyrosine-781 and serine 783.
In current search engines, the tryptic peptides generated would be generated from the first Methionine to the next positively charged residue and so on until the very last residue of the protein. If one would like to detect this protein in a sample and asses the presence of the mentioned PTMS, you would need to look for at least 2 phosphorylation sites per peptide and also check for S and Y phosphorylation. The search engine will then generate all possible combinations of SY single and double phosphorylate tryptic peptides to search for, which leads to exponentially increasing computational costs.
Since the protein is also cleaved by another protease in vivo, the 2 peptides before and after this site will not be accounted for as they do not end/begin after a positive residue. Since this is not a small protein, other peptides will probably still be detected, and the protein will eventually be identified.
I imagine a tool which would be used to generate the tryptic peptides as before, only accounting for the known PTM sites. In case of the ACE2 2 almost adjacent phosphorylation sites, this would lead to only 3 additional peptides (pY, pS, and pYpS). If the research question being asked is to identify novel phosphorylation sites, then only 1 phospho-site per peptide while looking for STY phosphorylation might already suffice, since the known ones will have already been accounted for. This can be applied to any combination of PTMs, massively reducing computational requirements. It is of course counterproductive to looking for PTMs in sterically inaccessible regions for example (e.g., hydrophobic core of the fold)
Databases of know annotated PTM sites of entire proteomes of many organisms are readily available. The tool could have a modular design in allowing the user to create a customized peptidome having any or all the following characteristics: trypsin/other enzyme used and/or accounting for known endogenous cleavage sites and/or accounting for known PTMs sites and/or accounting for natural variants.
I see a long list of advantages using this method and I would like to list the most important ones:
1. Identification of additional hits that could have been missed due to several reasons (e.g., tryptic peptides contain fixed modifications while not searching for these specific modifications due to computational resource limitation, or worse, small protein that would normally only yield in a single peptide that has 2 fixed modifications, one of which might be exotic)
2. Reduced computational time when trying to identify novel PTM sites
3. Lower false discovery rate since the peptidome used will be a much more closely related dataset to the actual sample composition than just a simple tryptic proteome and as a result newly identified spectra of interest can be more confidently assigned as the risk of artefacts is lower.
4. Single nucleotide polymorphisms can be analyzed analogously to PTM sites and would not result in exponentially larger search database.
5. More unique peptides could be assigned: If 2 proteins share a tryptic peptide, but one is known to be phosphorylated in this peptide but not the other, one could distinguish the phosphorylated peptide as having come only from one of the 2. In case of glycosylation this makes even more sense since some types of glycosylation only appear in a limited number of proteins, depending on their cellular localization
As the human proteoform project is taking on, maybe this would be the way of MS based proteomics to quickly catch up and help this project while advancing itself.
What are you thought on this? Are there any ongoing projects that would aim to do just that?
Relevant answer
Answer
To me, this debate seems somewhat reminiscent of the peptide-centric vs the spectrum-centric approaches.
Limiting the search space is generally a good idea for reducing FDR. Of course, if your peptide is not in your “limited” library you have no chance of identifying it. I see this as the biggest issue with this type of approach.
X!Tandem (and now other search tools) takes the approach that you do a broad initial search with few PTMs specified, then you broaden the PTMs once you have a smaller list of proteins to search. A neat approach in my view.
I’d be very careful with this source of information; “Databases of known annotated PTM sites of entire proteomes of many organisms are readily available.” I know of someone with a lot of de novo MS/MS experience who has undertaken an extensive manual review of phosphopeptides in the databases. The estimate (unpublished) is that around 30% are wrong. As tools progress and the amount of data increases, we look less at the raw MS/MS data. This is for very practical reasons, no one can manually verify 10,000 phosphopeptides, but we still need care when using this type of data.
Here are some papers that may be of relevance for you;
Lu, Yang Young, Jeff Bilmes, Ricard A Rodriguez-Mias, Judit Villén, and William Stafford Noble. “DIAmeter: Matching Peptides to Data-Independent Acquisition Mass Spectrometry Data.” Bioinformatics 37, no. Supplement_1 (July 1, 2021): i434–42. https://doi.org/10.1093/bioinformatics/btab284.
Searle, Brian C., Lindsay K. Pino, Jarrett D. Egertson, Ying S. Ting, Robert T. Lawrence, Brendan X. MacLean, Judit Villén, and Michael J. MacCoss. “Chromatogram Libraries Improve Peptide Detection and Quantification by Data Independent Acquisition Mass Spectrometry.” Nature Communications 9, no. 1 (December 3, 2018): 5128. https://doi.org/10.1038/s41467-018-07454-w.
Ludwig, Christina, Ludovic Gillet, George Rosenberger, Sabine Amon, Ben C. Collins, and Ruedi Aebersold. “Data‐independent Acquisition‐based SWATH‐MS for Quantitative Proteomics: A Tutorial.” Molecular Systems Biology 14, no. 8 (August 1, 2018): e8126. https://doi.org/10.15252/msb.20178126.
  • asked a question related to Proteomics
Question
4 answers
Hi everyone
I am looking to perform Protein extraction from Human Aortas to send for Mass spectrometry analysis. Anyone has previous experience with these tissues, and would be willing to share their protocol with me?
Thank you in advance for any help you may provide :)
Relevant answer
Answer
Hi Lara
Easy simple protocol:
Weight aorta sample : e.g. 100mg
Add tissue to FastPrep24 tube with Lysis Matrix D
Best will be if you can snap freeze the tissue in liquid N2 before taking the weight
Work always on ice (dry ice if possible)
Add Lysis Buffer (8M urea/100mM Tris-HCl pH 8.00/protease Inhibitors)
1mL Buffer/100mg tissue
Homogenize following Instruction on FastPrep24 Instrument
Centrifuge and transfer SN into new tube.
You can wash the beads once more with 1/2 Vol. of buffer and pool with first SN
With this lysate you can do what ever you want.
Protein Assay, run a gel, direct In solution digestion ...
Best wishes and good luck
Greatings
Natasha
  • asked a question related to Proteomics
Question
1 answer
What is the concentration of the surfactants used in the protein isolation, purification and crystallisation of proteins and what is the basis for selecting the surfactant concentration in the different steps in proteomics?
Relevant answer
Answer
Dear Subhrajit Mohanty sorry to see that your very interesting technical question has not yet received any expert answers. Personally, I'm not an expert in this field enough to give you a qualified answer. My suggestion would be to search the "Publications" and "Questions" sections of RG for relevant literature refernces and for closely related questions which have been asked earlier on RG. Moreover, please have a look at the following potentially useful review article which might help you in your analysis:
Successful amphiphiles as the key to crystallization of membrane proteins: Bridging theory and practice
This article has been posted by te authors as public full text on RG so that you can freely download it as pdf file.
I hope this helps. Good luck with your work!
  • asked a question related to Proteomics
Question
8 answers
Hello,
I have a very small knowledge in bioinformatics, and part of my research project is based on analysis of proteomics and metabolomics data. However, I am struggling to find some resources (webinars, courses, websites, ...) to help me get started with understanding and analyzing my data. I would appreciate it if anyone can give me some suggestions.
Thank you!
Relevant answer
Answer
Did you perform the experiment yourself? What kind of digestion? Do you have raw files? How versed are you with LC-MS/MS? Do you have access to any proprietary software e.g. Proteinscape or protein discoverer? Would you like to analyse the data yourself? For proteomics data MaxQuant is a wonderful and user friendly resource and a number of videos are available. Besides, the manual available on its website is quite self explanatory. For metabolomics data, MetaboloAnalyst is the analogous software. However, make yourself versed with the Jargon.
  • asked a question related to Proteomics
Question
5 answers
Hi,
As the protein buffer exchange is important for efficient protein immobilization. However, most times we lose some of the protein during the exchange process.
could we escape this step if the dilution factor is high, Ex; 50X or 100X? is there a reference for that?
Thank you in advance.
Best Wishes,
Waleed
Relevant answer
Answer
I never really had a major issue with protein loss by dialysis. Typically I lose most protein during column purification steps. Are you using desalting columns to exchange buffer or dialysis? If your yield is low it may just come down to switching to a different product. If you try diluting in your new buffer and have to re-concentrate with centrifuge filters, you will probably lose a lot of protein that way as well. I don't think there is any one method that is going to give you close to 100% yield, but there are many methods that should give you more than 90%. If this is not enough, then the issue may be with whatever approach you are using to express your protein. Maybe by optimizing that a bit more, your yield will increase enough that loss from buffer exchange is insignificant.
  • asked a question related to Proteomics
Question
2 answers
I am looking for a tool (online, R, Python, or otherwise) which I can use to highlight peptide sequences on the full protein sequence in a visually nice way for publications and presentations.
Extended description: In several of my bottom-up proteomics research projects, I have identified proteins of interest for a given condition/disease. Often, these proteins are activated/deactivated by cleavage (e.g. the complement system, coagulation system, angiotensinogen, etc.). Therefore, I commonly perform a peptide-centric analysis after the protein centric analysis, to identify changing peptides and then I manually map these to the protein sequence. I am looking for a tool to help me with this; where I can submit the list of peptide sequences and have these visually mapped to the full protein sequence of origin. Ideally, the tool should include known cleavage products (e.g. from UniProt KB).
Any advice is most welcome and thank you for your time.
Sincerely yours,
Tue Bjerg Bennike
Relevant answer
Answer
Maybe Peptigram can be of use for you?
  • asked a question related to Proteomics
Question
4 answers
I have done siRNA mediated knockdown of a low expressed protein in SKOV3 cell lines followed by proteomic analysis in biological triplicates. Proteomics was repeated three time. After retrieving the date I found that my desired protein(knockdown protein) in not present in transfected and even control (Non-transfected) group. However, I am getting bands of protein in western blot analysis. How can I justify my proteomics data.
Relevant answer
Answer
First, I would recommend to check the protein expression of your protein in the cell line. The chance to detect protein of interest with MS technique is low if it several copies per cell (WB usually more sensitive). Check how many tryptic peptides of your protein can be detected theoretically. Small mol. weight proteins can give you just too little tryptic peptide to claim protein Identification. Also it is a question how you performed proteomics analysis, was it a targeted analysis (MRM, PRM)? how sensitive and clean your MS? any potential PTMs on the peptides and so on? good luck
  • asked a question related to Proteomics
Question
3 answers
Hello,
I am looking to design a proteomics experiment looking at three treatment concentrations (Control, low-dose, high-dose) and two timepoints (24h, 48h) in an attempt to discover an unknown mechanism for lipid accumulation in THP-1 macrophages. I have never stepped into the omics world before so I thought I would start by asking:
What do you know now that you wish you had known when you started?
Relevant answer
Answer
Hi Braeden Giles
Mass spectrometry is a very sensitive and accurate method to determine the precise molecular weight of a protein.
Because it is very sensitive and is amenable to rapid processing of many samples, it is becoming very popular for determining the different proteins present in complex samples (e.g., the proteins in a given cellular organelle, separated by two-dimensional gel electrophoresis), in what is known as a proteomics analysis.
Techniques have now been developed by which proteins separated in two- dimensional gels can be digested within the gels using endoproteases and then injected directly into a mass spectrometer for analysis of the resulting fragments.
Best
  • asked a question related to Proteomics
Question
9 answers
In fact, we know that both active transport and facilitated diffusion can using the carrier protein. And in the processes, there are involving conformation change, that change energy depends on a)facilitated diffusion→ligands disruption of the carrier intermoleuclar forces, b)atp as energy
however it is interesting that if free collision of ligands to receptor can lead conformation, then why or what molecular mechanism driven that in the case of against concentration gradient have to depends on the atp(atlases case) but not the ligands receptorinteraction changing the interaction?
to ask in the other way, can I engeering a new protein that they are able to transport again concentration gradient but no need atp /cotransporter, only depends on the channel interaction and free flowing of the ligands (collision frequency)? thank you
Relevant answer
Answer
If I understand your question correctly, both facilitated diffusion (FD) and active transport (AT) are both mediated by transmembrane proteins. In the FD the driving force for the conformational change in the protein is the concentration gradient (chemical potential) of the molecules being transported. In AT, the driving force is the hydrolysis can either be a nucleotide phosphate OR the chemical potential of a co-transported species. This co-transported species can be the proton motive force PMF, but can also be Na+, K+, Ca+ ion concentration gradients. Most of AT is by the latter mechanism, rather than nucleotide hydrolysis. Note, there is also a minor amount of FD going on with membrane-soluble small molecules (e.g., quinones, etc) that are not proteins, but the driving force for the transport is the chemical potential.
I am not particularly fond of the AT/FD terminology, which I think is now archaic based on historical lack of knowledge about the wide variety of underlying mechnanisms, but they are descriptive of the driving forces. I would prefer the use of coupled transport (CT) instead of AT as this allows for more mechanisms to make the overall Gibb's free energy change of the transport negative (exergonic), including the conformational change in the transmembrane protein.
When thinking about the protein-facilitators involved in both FD and CT, you have to consider these gated porin proteins which only open on one side of the membrane at a time. It is the Gibb's free energy change (either chemical potential or the molecule or a co-transported species, or nucleotide hydrolysis) of the transport that drives the structural change in the protein allowing the pore opening to shift from one side of the membrane to the other. The transported molecule(s) bind to a binding site interior to the pore.
There is a lot of thermodynamics and math behind all this, which is poorly described in the biochemistry literature, probably because biochemists typically don't have a lot of thermodynamics background. I have a Chapter in my up-coming Biological Engineering textbook that covers all this.
  • asked a question related to Proteomics
Question
3 answers
I have generated spheroids (UN-KC6141, pancreatic cancer cell line) and want to do proteomics.
Anyone who has a protocol for this procedure?
Relevant answer
Answer
Hope all the best for you.
please don,t hesitate from contact via email, if more help needed.
Yours truly
  • asked a question related to Proteomics
Question
3 answers
Hi all,
I have been working on optimizing lysis conditions to do whole proteome lysis from liver tissue and have a head-scratcher. Using the BioRad detergent compatible BCA analysis kit, I get a woefully low estimation of protein extracted when compared to doing a mass balance (weighing empty tube, liver, then remaining pellet after extraction)... I've washed the liver tissue as much as possible to remove blood (non-perfused at harvest) and the samples aren't bloody looking. Does anyone have any suggestions of expected protein extracted per wet liver weight? If I know that, I can at least have a better idea of which number I should use (BCA or mass balance).
many thanks
Relevant answer
As a very general rule of thumb, the protein yield after tissue extraction in SDS or urea buffers with sonication/bead beater for total cell lysis, including organelles (whole proteome extraction) will be approximately 5-10% of the initial wet weight. So from a piece of tissue that weighs 100 mg, one might expect 5-10 mg of protein. This varies of course depending on the type of tissue being processed, the stringency of the lysis/extraction reagents and process (are you pulverizing the frozen tissue first?), whether the tissue is dehydrated, and the volume of buffer used for lysis.
  • asked a question related to Proteomics
Question
3 answers
Dear all,
we are interested in label-free quantification of shotgun proteomics experiments using the Waters SYNAPT G2-S HDMS instrument.
Does anybody have any experience of such data being used with MaxQuant?
What other freeware could be used for such application?
We are not sure if MSe mode or Survey mode would work best.
Thanks for your help!
Relevant answer
Answer
For future interested users, this publication has been useful
  • asked a question related to Proteomics
Question
3 answers
Hi all,
I want to learn how to work with these databases? I do not know how to learn them step by step, and there is no instructional video.
-Genevestigator
- ProteoCloud
-PeptideAtlas
- Chorus
Thanks for all your help.
#molecular_biology
#proteomics
#functional_genomics
Relevant answer
Answer
First, find the problem, the learning will be added to your mindset by default. Anyhow, youtube, Coursera or even some text (pdf) based tutorials are already available on the internet.
  • asked a question related to Proteomics
Question
5 answers
Hi All,
I am using zeba desalting column to purify my protein + plasma sample in the beginning step of protein enrichment procedure to get rid of all unwanted stuff. After performing desalting process, I am going through the enrichment and digestion procedure but on LC-MS my peptide peak shape getting poor as the more number of injections injected. Initially peak shape is perfect but after injecting about 30 to 40 samples, chromatography getting poor. Earlier, samples prepared without zeba desalting have not shown any poor chromatography. Anyone have any idea about troubleshooting?
Thanks.
Relevant answer
Answer
Kindly, Check the estimated yield of sample in the eluate of desalting column.
  • asked a question related to Proteomics
Question
1 answer
I have some interest in the interaction between protein A and B but I barely know about proteomics so I leave the question here.
To specify the exact interaction sites, I made four site-directed mutated plasmids having a GST tag using the quick-change method. The mutated sites are on the cold shock domain of protein B. Because I read this phrase "systematic alanine scanning mutagenesis has revealed that the substitution of an amino acid residue by alanine in these hot spot regions lowers the binding affinity by at least 2 kcal/mol (Bogan and Thorn 1998).", I changed every mutagenesis site into alanine.
And then I did a GST pull-down assay after co-expression of MYC-A and GST-B (treated with RNase, DNase, and MNase). This is the question. I could not understand the results I got from this experiment. The affinity between protein A and mutated B(all four mutations!!!) is so much higher(>100 folds) than that between protein A and wild-type protein B. They interact much stronger via these mutated sites. Do you have any idea what it is? And can it be a clue for finding exact interaction sites?
Relevant answer
Answer
Reading about biotechnology database and bioinformatics directory in this article may help you find your way through your research
This article published by Stanford university
  • asked a question related to Proteomics
Question
3 answers
Hi all,
I was wondering if anyone knows-
which statistical test I should use in order to find whether a sample is an outlier in my proteomic data? It's obvious when looking at the PCA, but how should one calculate this?
Many thanks for your help!
Relevant answer
Answer
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.
  • asked a question related to Proteomics
Question
3 answers
Hi everybody. I have been in metabolomics 10 years and proteomics 20 years. However I feel confuse today that I am not going anywhere. It is endless questions and endless work to do? Is it good or bad for my future? Have I going wrong direction on the beginning? Today, every manuscript you read it has something to do with programming. Is it future? Should change my career as soon as possible?
Relevant answer
Answer
Having done proteomics and metabolomics research for 25 of the last 35 years, I feel your pain. I can only leave you with the following paragraph from Chapter 1 of my biological engineering textbook (hopefully to be published this year).
"For the last three decades, biological science has been dominated by those espousing: 'In ten years time, those with a hypothesis-based approach to science will be equivalent to those who believe in the Flat Earth theory.'[1] To whom I counter with Huxley’s argument that won the Great Evolution Debate, “Six monkeys with typewriters … and an infinite amount of time will collectively produce all the great works of man” (June 30, 1860). Infinite time and resources do not exist in the real world."
[1] Gannon, F. (2000) “Back to Darwin?”, EMBO Rep., 1:373.
To the best of my knowledge, there has been 1 new commercially-validated biomarker used for clincial diagnostic purposes discovered with all the effort in genomics, proteomics, and metabolomics combined, since 1990. Yet, there has been an untold amount of white noise generated in the scientific literature. The conclusion of every paper that I've ever read has been "more study is needed". For every new biomarker one paper 'discovers', I can find another that shows it wasn't significant. Great for government funding agencies. Great for Universities, Great for tenure committees. Great for scientific publishers. But, did all that money and effort really serve society? Steve Naylor (former CSO of Beyond Genomics) once told me, that they spent $22M studying 300,000 functional genomic, proteomic and metabolic features in 40 mice to prove their platform worked. When they went to sell the pharmaceutical companies on their biomarker discovery platform, the consensus was "$22M are you F___ing nuts".
Don't get caught in what economists call "the sunk-cost fallacy". Find something that is causing others real pain in their lives for which you have a better mouse trap and can support yourself doing that. Always remember that government is the funder of last-resort. If what you do was really providing a net benefit people, they would be paying you for it directly. You wouldn't need government to take the money to pay you for it from others by force. I'm retired now, with enough in the bank to live out my life comfortably. I've already been 'cancelled', hence the forced retirement, so I can afford to say it they way I see it. Your decision, however, is yours. Either way, expect that decision is going to get you 'cancelled' by someone. The opinions of others are like arses. Everyone has one and they all stink. You have to do what you can live with.
  • asked a question related to Proteomics
Question
6 answers
Hi all,
Are there any strong books or tutorials on the concepts underpinning manual inspection and editing of alignments garnered by our alignment algorithms? Applying to both pairwise and MSAs, and I know the difference is perhaps stark between the two, though not sure.
Warm regards,
Michael
  • asked a question related to Proteomics
Question
5 answers
Hello everyone,
I am trying to sort cells using FACS and the sorted cells will be processed for downstream study, sequencing, proteomics, or metabolics, for example. I am gonna use two reporter lines to tell the given cells from each other, tdTomato and GFP. But now the problem is that our BD Aria II doesnot have a 561 laser line, so tdTomato-positive cells cannot be sorted out, I am trying to use anti-tdTomato Ab that is conjugated to 633 to stain the cell suspension, but I donot know if it can make it. Usually, the Ab is specially produced for flow cytometry, and also it only recognize the antigen on the cellular membrane. tdTomato is expressed intracellularly, I have to use a blocking buffer containing detergent to puncture the membrane, Trition-100 for example, and let the Ab enter into the cells and bind, if in that case, it means there must be intracellular content efflux, which might cripple the downstream experiment, proteomics and metabolics for example.
Anyone can let me know how to deal with this in my case.
Thanks a lot !
Hao
Relevant answer
Answer
Hello, Lukas, I hope everything goes well with you. Now I am trying to estimate cell viability using PI through flow cytometry, no sorting required, I just want to see how many cells are survived, do I need to set a control that is treated using DNase I. Thanks a lot !
  • asked a question related to Proteomics
Question
3 answers
in Western blotting, we use loading controls such as alpha tubulin and beta actin are used as loading controls to normalize samples. Can this method also be applied to normalize results obtained for shotgun proteomics?
Relevant answer
Answer
In immunoblotting experiments, we use housekeeping proteins to verify the sample preparation reproducibility and for showing the fold change is a result of biological variation. At this point of view, we assume the levels of the abovementioned structural housekeeping proteins are the same for purposed samples. On the other hand, recently total protein normalization is a more reliable approach to test this hypothesis rather than housekeeping normalization. According to the question you asked, a certain and single answer cannot be given I guess. There are several ways to perform absolute and relative quantification at shotgun proteomics at mass spec. To validate the sample preparation, spiked in peptides or proteins and/or adding digestion control is good options to implement. With these approaches, you can both validate ms and sample preparation performances. I am sharing a couple of products to be used below. If you trust enough your housekeeping proteins they can be used for this purpose but I am not sure resulting data is acceptable or not for the journals. You have to prove the analytical variations by spiking proteins and or peptides besides the biological internal controls.
Pierce™ 6 Protein Digest, equimolar, LC-MS grade 88342- 100 pmol
Pierce™ Digestion Indicator for Mass Spectrometry, - 84841 – 10 μg
6 x 5 LC-MS/MS Peptide Reference Mix– V7491– 50 μl
Agilent peptide standards, 5190-0583, 10 peptide standard lyophilized, 71 ug in 2 ml vial.
Emir…
  • asked a question related to Proteomics
Question
4 answers
We are using HRP conjugated secondary antibody for blotting and we employ DAB as developer. Some times the bands appear very much intense (dark brown) whereas sometimes the bands don't appear intensely with same protein concentration. We have changed all the stock buffers as well as tried a new vial of antibody but the problem persists. Why does this happen? Is it due to variation in the HRP activity?
Relevant answer
Answer
@Jatin Chadha the stock concentration of antibody from the manufacturer is 0.5 mg/ml in PBS with 50% glycerol. We are using at 1:15000 (i.e. 1 microlitre in 15 ml of TBST) or roughly 500ng per blot with 1 hour incubation at RT. Prior to antibody addition we block the membrane with 3% skim milk powder in TBST for 1 hr at RT in most of the cases or for O/N at 4 degree C.
  • asked a question related to Proteomics
Question
1 answer
We use R&D Proteome Profiler Rat Cytokine Array for detection of multiple cytokins in tissue samples. The standard protocol utilizes streptavidin-HRP with a chemiluminescent detection reagent that is not specified in the product documents. Does anyone know, what is the two reagents provided to the kit (chemi reagent 1, chemi reagent 2)?
Have anyone tried another detection system for visualizing (either chemiluminescent or other) the dots?
Relevant answer
Answer
We use R&D Proteome Profiler Human Cytokine Array for screening different cytokins. Chemi reagent 1 is hydrogen peroxide and Chemi Reagent 2 is a stabilized luminol.
For your question about using different detection reagents,
Yes, we used Thermo Scientific Pierce ECL and it works perfectly well.
  • asked a question related to Proteomics
Question
6 answers
I studied the whole-cell protein profile in cyanobacteria undergoing abiotic stress. I have two questions regarding the data.
1. Is it mandatory/preferable to submit whole-cell proteomics data to a repository/database before publishing?
2. If it is, what is the best repository/database especially for cyanobacteria?
Relevant answer
Answer
repository deposition of the raw proteomics data is required for most journal as a way of validating the authenticity of the data in the manuscript. It will also increase the global publicity of your manuscript
  • asked a question related to Proteomics
Question
5 answers
A primary look at imputation methods feels like we are just inventing values to make the data fit.
e.g.
For data where we impute to improve the replicate clustering: aren't we forcing the replicates to agree?
For data where the protein is missing but we impute some kind of probabilistic value, what if the protein is actually absent?
For data where the absent values are non-random and non-ignorable, do we know the technical cause of missing values to impute?
How do we know whether our imputation has made the data better or caused us to introduce artefacts?
Relevant answer
Answer
I hope all your queries get answered with these articles:
Thank you.
Varsha
  • asked a question related to Proteomics
Question
5 answers
Hello dear fellow scientists,
I would like to ask some basic naive questions:
1) when scientists perform a transcriptomic study, lets say to compare a mutant to a Wild type plant, they tend to look at the genes that are at least 2 times more or two times less expressed between the two samples, why not all genes that are differentially expressed between the two genotypes? is it because it is more reliable ?
2) Usually when you perform a transcriptomic and a proteomic study (on the same sample and same conditions) you only find a low number of genes that show the same expression pattern (up-regulation or downregulation) between the two experiments, why ??
I did a transcriptomic and a proteomic study on a mutant and I found a small overlap between the differentially expressed genes and the differentially expressed proteins,
I mean its not surprising overall but I can't think of an explanation,
is it related to the degradation of transcripts ? post-translational regulations ?
I hope my questions are clear..
Sincerely
Relevant answer
Answer
Yes indeed... Polyadenylation controls many things, but mainly the stability of the transcript. It is well known in mammals that for instance those proteins involved in signal transduction have the tendency to harbor weaker polyadenylation signals, which conditions the stability of the mRNA and the protein levels.
  • asked a question related to Proteomics
Question
3 answers
Could someone point me in the right direction as to which tool will allow me to analyse a proteomics dataset and filter the list of proteins in it based on their function (e.g. proteins related to cytoskeleton, metastasis etc) yielding a list of such proteins/ network?
Thanks!
Relevant answer
Answer
Uniprot/Gene Onthology. And if you want to go deep in information, there is also KEGG pathway database.
  • asked a question related to Proteomics
Question
4 answers
Data extracted from Nano-LC-MS,
I want to submit the mass spectrometry proteomics data to the ProteomeXchange Consortium via the PRIDE
Relevant answer
Answer
Thank you for your presence attention to all answers
  • asked a question related to Proteomics
Question
2 answers
We have used Agilent nanoLC QTOF MS/MS system for proteomics studies. The format of raw data file is .d and searched files from Spectrum Mill software are in .ssv format. How we can convert these searched file format into mzIdentML or mzTab ??
Suggestions or answers will be highly appreciated.
Relevant answer
Answer
We will try to do. Thanks for your reply.
  • asked a question related to Proteomics
Question
5 answers
Hi all,
I would appreciate any experience-based input on free software tools to analyze proteomics data. I have read the theoretical basis but no hands-on experience...
Thanks in advance
Relevant answer
Answer
Why don't you try some Python pre-existing code?
  • asked a question related to Proteomics
Question
5 answers
Hello,
I need to deposit our mass spectrometry proteomics data to one of the good proteomics repositories acceptable for publication.
I found many published papers deposited their data to the ProteomeXchange Consortium via the PRIDE partner repository.
I am completely new on doing this and quite confused about what is the most simple and straightforward way for depositing the data to ProteomeXchange Consortium via the PRIDE.
I would highly appreciate if you could advise me in this regard. What is their requirements for mass spectrometry proteomics datasets? Which tools is the best to do it? How long does usually it take to get an identifier?
Also, can we only deposit our proteomics datasets in PRIDE? and not to ProteomeXchange?
Many thanks for your great help and advice.
Best wishes,
Farah
Relevant answer
Answer
Farah Farah As already you have been answered, the upload process is very easy once you download the tool. You will have the option for partial submission or complete submission. You can opt for either and if chosen partial, try to make it public once your article is accepted for publication.
The process of upload will depend upon internet connectivity!
  • asked a question related to Proteomics
Question
2 answers
Dear,
I am working on cell cultures and labeling nascent translated proteins with methionine analog - AHA. Then I perform "click reaction" on cell lysates to tag freshly translated proteins with biotin. Can anyone advise me on what type of magnetic dynabeads is best for pulling down full AHA-biotin labeled proteins? I have successfully used M-270 dynabeads to pull down tryptic AHA-biotin labeled peptides but for full proteins I have good enrichment only for small <35 kDa proteins. Are some other dynabeads like C1 streptavidin better for full proteins? What can I do and what types of beads are best for pull-down of larger proteins. All suggestions and advice are welcome.
Relevant answer
Answer
Good Morning,
We have 4 different types of streptavidin coated Dynabeads™. They differ in size, binding capacity, and surface charge. These four beads are based upon 2 different chemistries, tosyl based chemistry and carboxylic acid based chemistry. Streptavidin is covalently coupled to these functional groups in order for biotin to bind. We have 2 medium sized 2.8 µm, one which is based upon a Tosylactivated bead meaning that it is a hydrophobic bead (Dynabeads™ M-280 Streptavidin, 112.05). The other one is based upon a carboxylic acid bead meaning that it is hydrophilic in nature (Dynabeads™ M-270 Streptavidin, 653.05). Then we have a 1 µm alternative which is called MyOne™ (like all other 1µm Dynabeads™) one based upon a Tosylactivated (Dynabeads™ MyOne™ T1, 656.01) and another based upon a carboxylic acid (Dynabeads™ MyOne™ C1, 650.01) bead meaning that they will differ in hydrophobicity and binding capacity.
Dynabeads™ M-280/T1 Streptavidin works in applications such as immunoassays, purification of DNA/RNA binding proteins, protein purification, cell isolation etc.
Dynabeads™ M-270/C1 Streptavidin works in the same applications such as nucleic acid assays, but also in protein isolations.
For you application - the use of a MyOne (1µm) bead would increase the capacity, and for the protein application I would suggest to test either the M-280 Strep or the T1 Strep bead. We have also automated the IP protocol on the KingFisher Flex and DuoPrime instruments for a more or less walk away solution.
good luck
kind regards
ketil
  • asked a question related to Proteomics
Question
4 answers
Greetings! I am looking forward for Liquid nitrogen free method of Protein isolation (from leaves) for Proteomics. I intend to follow TCA method for the purpose. It would be a great favour if someone guide me in this regard. Also, guide me how to clean mortar and pestle to avoid protein contamination while crushing different samples. Thanks in advance!
Relevant answer
Answer
Hi!
I used lyophilization followed by SDS, DTT protein extraction. It worked perfectly good on my leaves samples. You can check it on my paper
  • asked a question related to Proteomics
Question
5 answers
How long can fusion protein be stored at -20°C? If you need your protein samples for longer periods of time, is there any trick for storing them at -80°C?
Thanks in advance.
Relevant answer
Answer
Malcolm Nobre , I don't see how freezing protein droplets in liquid nitrogen makes sense. You need to store the protein protein in a tube to put it into the minus 80 freezer. So what's the point? What's innovative about writing a publication about flash freezing proteins in tubes in liquid nitrogen? Doesn't everyone do that!?
  • asked a question related to Proteomics
Question
3 answers
Hi
In past I was using MASCOT to search, identify and explore the proteomics data. I am looking for any windows based free software to analyze my big proteomics data files mostly in mgf format.
Relevant answer
Answer
Thanks everyone. I have downloaded the Maxquant...let's see how it goes....
I am looking for both quantification as well as identification...moreover looking for PTMs too
  • asked a question related to Proteomics
Question
1 answer
I have been working on pancreatic islet isolation and have no prior experience with pancreatic islets. Our aim is to use the islets for omics studies. I would like to know the best strategy for homogenizing the freshly isolated islets for proteomics studies (buffers, time, temperature, etc.) if someone already has prior experience with them.
Relevant answer
Answer
You can try this buffer: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% TritonX-100 plus proteinase inhibitor cocktail (Note there is no SDS in this buffer). Relevant literature: https://diabetes.diabetesjournals.org/content/69/8/1723
Good luck!
Anoop
  • asked a question related to Proteomics
Question
2 answers
Plant research (Genomics, epigenetic, proteomics)
Relevant answer
Answer
@Desirée
Please specify from which origin you are going to do RNA sequence? If plant species, you may contact through message .
  • asked a question related to Proteomics
Question
2 answers
We're supposed to delete rows that have blank/Zero as LFQ intensities and those that have <0.75 individual localization probabilities for all replicates in one or both groups. (If they have average loc. prob. <0.75 they will be deleted prior to this). My question is how many replicates in a group must have below threshold values in order to be deleted. 50%? If in a group with 6 replicates, there's only one replicate that has loc. prob more than 0.75, should we retain it?
Relevant answer
Answer
For LFQ, at least 3 replicates for each group is essential. Not sure why you use <0.75 individual localization probabilities. For global LFQ discovery experiment, 5% of the total identified proteins were selected as the DAPs, and for cut-off value determination.
  • asked a question related to Proteomics
Question
2 answers
Hey guys,
to gain access to proteins inside of Lactobacillus casei and L. plantarum vesicles and to perform LC-MS/MS, I want to establish a lysis protocol. The protein concentrations before and after the lysis are determined with a BCA assay.
I already tried Triton X 100 buffer (4h, on ice) and RIPA buffer, but the results were not reproducible or it didn't work at all (same protein concentration before and after lysis).
I also tried a sonication bath after incubation with buffer and 30s with 50% intensity digital sonifier. Lyophilization and solving in Methanol didn't work either.
Did anyone have similar problems? Do you know a working lysis protocol for these (seemingly very stable) EV's?
Relevant answer
Answer
You can try to use 6M urea, 2M thiourea, up to 5% SDS for EVs lysis. And after extraction, I do suggest to running an SDS gel for checking the EVs' proteins complexity and do quantification by gel.
  • asked a question related to Proteomics
Question
4 answers
Hi everyone,
I'm currently delving into proteomics head first, which is entirely new to me. My collaborators will be carrying out tandem mass tag spectrometry with fractionation on my samples (cases versus controls, tissue is postmortem brain tissue) and will be sending processed results my way, which include # Peptides, # Unique Peptides, values scaled to QC, % CV, abundances, normalized abundances. I'm interested in case versus control differences so what would be the best analyses to do? I'm only familiar with RNA-seq analyses, so any workshops, youtube tutorials, tips and advice would be GREATLY appreciated!
Thanks,
Marina
Relevant answer
Answer
You can impute the normalized data in Perseus and do this kind of analysis (Cases versus controls). If you have any questions you can watch the MaxQuant Summer School videos.
My reference:
Perseus link:
MaxQuant Summer School:
Any questions send me a direct message.
Best regards,
Leite
  • asked a question related to Proteomics
Question
4 answers
Would proteomics studies matter in drought tolerance screening for legume crop accessions?
Relevant answer
Answer
Hallo,
Proteomics research related to the effects of water deficits on plants might be productive, but only if done using the strictest methods to control and measure the water status of plants. May I suggest reading:
Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities
David W. Lawlor
Journal of Experimental Botany, Volume 64, Issue 1, January 2013, Pages 83–108, https://doi.org/10.1093/jxb/ers326
Which explains the problem and a way of dealing with it.
Regards
  • asked a question related to Proteomics
Question
3 answers
Hi. Recently I am working on proteomic data analysis, and I need to know how many times A protein is expressed more than B protein (mass or copy numbers). I know there are multiple ways of proteomic methods to compare between-sample levels, in other words, the relative number of one protein in two or more samples. I'm wondering whether can I quantify the relative number of protein A over protein B in the same sample. If this can be done, how to deal with the proteomic results (e.g. the intensity matrix of peptides or proteins) to get the relative number I want? Thanks!
Relevant answer
Answer
The most accurate thing you can do is SRM using heavy peptides. This will give you absolute quant for each peptide. That said, there are a number of publications showing it is possible label free;
J.R. Winiewski, M.Y. Hein, J. Cox, M. Mann, A “proteomic ruler” for protein copy number and concentration estimation without spike-in standards, Mol Cell Proteomics. (2014) mcp.M113.037309. https://doi.org/10.1074/mcp.M113.037309.
Schubert, Olga T., Christina Ludwig, Maria Kogadeeva, Michael Zimmermann, George Rosenberger, Martin Gengenbacher, Ludovic C. Gillet, et al. “Absolute Proteome Composition and Dynamics during Dormancy and Resuscitation of Mycobacterium Tuberculosis.” Cell Host & Microbe 18, no. 1 (July 8, 2015): 96–108. https://doi.org/10.1016/j.chom.2015.06.001.
He, Bing, Jian Shi, Xinwen Wang, Hui Jiang, and Hao-Jie Zhu. “Label-Free Absolute Protein Quantification with Data-Independent Acquisition.” Journal of Proteomics, March 14, 2019. https://doi.org/10.1016/j.jprot.2019.03.005.
Sánchez, Benjamín J., Petri-Jaan Lahtvee, Kate Campbell, Sergo Kasvandik, Rosemary Yu, Iván Domenzain, Aleksej Zelezniak, and Jens Nielsen. “Benchmarking Accuracy and Precision of Intensity-Based Absolute Quantification of Protein Abundances in Saccharomyces Cerevisiae.” PROTEOMICS 21, no. 15 (2021): 2170095. https://doi.org/10.1002/pmic.202170095.
  • asked a question related to Proteomics
Question
6 answers
Some studies say that the Random forest method could be the best. But I'd like to get more opinions since many people seem to be using many different methods. It would be nice if someone could provide any resources for carrying out the methods too (Tutorials, R code, etc)
Relevant answer
Answer
Hi Joshua
You don't need opinions as you can solve the problem without relying on other people's preferred methods of imputation.
The process is: take your full data and remove (say) 2% of it at random. Then try several imputation methods to replace those values. Compare predictions to known observations. The method with the smallest error is the one to use when imputing your genuinely unknown values.
  • asked a question related to Proteomics
Question
3 answers
Hi. Since missing value imputation is determined by nearby data, should we separate control and treatment groups and perform MVI separately for each? Context: This is for mass spectrometry data.
Relevant answer
Answer
Yes. It is better to calculate MVI for separate control and treatment groups.
  • asked a question related to Proteomics
Question
5 answers
Hello!
I am working with some materials that require desalting of the peptide samples prior testing. My samples contain a salt of potassium. With a standard sample, I would use R3 or C18 resins with low pH solvents for desalting (Formic acid + MeCN, for example). However, I am working with a modification that is unstable at low pH but completely stable at high pH.
Can you de-salt peptide digest using C18 or similar with basic pH solvents as if you were doing high pH RPLC fractionation? In our lab we always desalt prior high pH fractionation but we do it with acidic conditions. But with high pH, would the peptides be retained in the C18 stationary phase? And if it's possible, what kind of solvents would be good for it? (Keep in mind that the peptides are analyzed by LC-MS/MS).
Thank you for your advice.
Ignacio
Relevant answer
Answer
Organosilica C18 and completely polymeric ones can retain most of the acidic and even basic peptides at high pH. Besides net charge, hydrophobic AA composition and total HPLC index of the peptides can indicate whether the target peptide may retain much or less by using high pH conditions. ACN and water modified by NH4OH are ok for MS. What kind of C18 properties do you have with your purposed material as zip tip? pH stability, backbone structure, porosity/pore size etc...as you suspected not every C18 is the same.
  • asked a question related to Proteomics
Question
8 answers
Hi!
I am experiencing some difficulties in preparing brain tissue for proteomic analysis - most likely due to high lipid content that interferes with proteomic data acquisition.
We need to isolate proteins in their native state including larger molecular weight complexes and protein aggregates. This means that we are limited to bead beating in native lysis buffer (1 mM MgCl2, 150 mM KCl, 100 mM HEPES, pH 7.4) and a very mild centrifugation step (800xg, 5 min) before using the supernatant for tryptic digest, C18-cleanup and HPLC-MS.
However, I commonly observe column clogging or ion suppression using these samples, which I suspect is due to the presence of contaminating lipids.
Does anyone have a good protocol to remove lipids without denaturing proteins or for removing lipids after the tryptic digest?
Any advice is highly appreciated! Many thanks in advance!
Relevant answer
Answer
Dear Norbert,
Your question is a challenging one!...and as you say the clogging and ion suppression issues in the LC-MS analysis may be due to the presence of lipids in your sample.
As removal (extraction) of lipids typically involves the use of organic solvents this will also denature your protein. So in my opinion, it might be an option to remove lipids after the tryptic digestion step (and assuming that lipids do not restrict the protein digestion).
You can find commercially available SPE columns (Supelco) designed to retain (phospho)lipids from biological samples - SPE Hybrid-Phospholipid.
Hope this helps,
Ana
  • asked a question related to Proteomics
Question
4 answers
Its hard to detect amyloids even in brain tissue of Alzheimer patients using untarget proteomic methods. I am curious of what kind of protein is hard to detect by mass spectrometry, whether there are commonalities in their structures?
Relevant answer
Answer
Also hydrophobic regions (like transmembrane regions) which do not harbor charged residues (mainly positive charges, important both for fragmentation) are very hard to analyze with MS.
  • asked a question related to Proteomics
Question
5 answers
I'm conducting proteomic research on a complex in plasma. I found when I set the criteria as at least 2 unique peptides, lots of low molecule proteins (~10 kD) were excluded despite they have relatively high PSM. In my experience, these proteins have already been validated composing this complex.
I am wondering if I could comfirm all unqiue peptides of a protein in theoretically? Is there any database I can seek? Or for some low molecular proteins, only 1 unique peptide enough to identify their existence in proteomic study? P.S. These proteins were usually members of a superfamily.
Thank you.
  • asked a question related to Proteomics
Question
2 answers
Hi,
I am looking for an active and productive collaboration with experienced researchers in the area of analysing (wet lab validation and bioinformatics) human genome, transcriptions, proteome, metabolome, single cell omics etc. for various human traits and disease related research.
Please contact at dr.barh@gmail.com
Relevant answer
Answer
Let me know if you are interested to collaborate with my lab, Eminent Biosciences
  • asked a question related to Proteomics
Question
3 answers
I have to use mass spectrometry proteome data (analyzed data from Maxquant) to carry out post-translational modification analysis using Proteome Discoverer software. I'm searching for a proper workflow for this purpose. Any suggestions/links to useful sources, etc. would be very welcome.
Relevant answer
Answer
Hi Joshua,
when you already have analyzed data from MaxQuant a search with Proteome Discoverer is (to my knowledge) not possible, as this programme is using the machine raw files for analyis. However, MaxQuant has an option including PTMs and also gives you the information on their position. You can process your MaxQuant files with Perseus, to analyse your PTMs. If you want to use PD for PTM analysis, then you have to use the raw files and you include your PTMS in the search engine tab.
  • asked a question related to Proteomics
Question
9 answers
I am interested in how plants shape microbial metabolism in the soil and I was wondering if we could extract "soil proteins" as we do for genomic DNA extraction from soil and perform proteomics to understand how plants shape soil metabolism? If it is possible, would you suggest some references to read and follow?
Relevant answer
Answer
Thank you all for your kind suggestions!
And Arnab Banerjee I appreciated your insights, I too had similar ideas. Though, It seems like the transcriptomic approach is being used and is more widely accepted. In fact, I tried extracting proteins from soil and ran SDS-PAGE according to Benndorf et al. (2007) and Heyer et al. 2019's work and the results were visible but I had many questions popping up from my head as you have mentioned above.
Anyways, thank you all again for your kind suggestions.