Hello, I am new to proteomics and we are designing some experiments to look at proteomic profiles from cell lysates. Our previous MS method needed a more simplifed mixture of proteins, so we had been manually separated the lysate using sequential molecular weight cut-off filters (10 kDa, 30 kDa, 50 kDa, and 100 kDa, definitely not ideal!). We are hoping to move to using an LC-Orbitrap method for analysis and my understanding is that using an LC MS/MS technique (starting with reverse phase LC that is coupled with an orbitrap fusion tribid) we may only need to minimally separate the sample prior to LC. I'm wondering if anyone has worked with the Orbitrap and/or with complex cell lysates and can advise whether we could simplify our pre-fractionation methods prior to the LC step?
Hopefully I have been clear! Thanks for any ideas and suggestions
Hi Marianne,
first of all, congratulations to the Fusion. I would like to have one as well. You should already get relativley good coverage without any pre-fractionation. So, just an in-solution digest or a 1-fraction in-gel digest. Of course, coverage will be improved with increased fractionation.
The following article might be helpful (the one hour yeast proteome):
http://www.ncbi.nlm.nih.gov/pubmed/24143002
Best
Florian
Great, thanks for all these ideas! Florian, I wish the Fusion was mine! We are contracting out to a Proteomics Center in British Columbia. So we get charged per sample, and that's one of the reasons I would like to pre-fractionate as little as possible, but still get good yield. [The other reason is I want to minimize sample handling and potential protein loss!]
We analyse several whole cell lysates performing pre-fractionation of peptides after tryptic digestion. The number of fractions that you "need" depends of the aim you are analysing your sample. If you are going only to identify, maybe 6 fractions with a MCX-cartridge it is enough to obtain near to 8000 proteins at 1% FDR (in 2 hours of analysis each). But if you are going to quantify with an isobaric labelling, this number of fractions could not be enough to get a proper quantification due to interferences with isobaric peptides.
Adding to what Fernando said: assuming you are talking about peptides analysis, you have two main procedures.
a) cation exchange fractionation (sequential elution with buffers at different pH). This is the method Fernando wrote about and it is the most commonly used for whole proteome analysis of tissue, cells and plasma proteins.
b) simple cleanup with packed tips (C8 or C18 resins). this approach does not increase the number of samples (therefore it saves up time), but the samples are way more complex. In addition, high abundant proteins generates peptides that can mask other analytes of interest.
It is also true that, for a "first try" this might be the method you want to chose, for it is very simple. In addition, the Fusion has several features that can help with sample complexity.
Detergents: Pawel suggestion is a key point for a successful MS analysis. Strong detergents are very powerful in dissolving proteins with high hydrophobicity. Unfortunately, they KILL the ion signal. If you chose detergents such as SDS, Triton X and so on, you need to cleanup your sample
I would suggest you two ways:
- FASP: if you have a relatively high amount of sample, you can use the filter aided sample preparation approach, which allows the use of SDS. Note: peptide recovery has been evaluated at 50%.
- cleavable detergents: a novel approach is emerging nowadays, in which detergent can be eliminated from sample by treatment with acid (e.g. formic acid in the mobile phase, which I expect you will use to reconstitute your sample prior to MS analysis) or UV light. Waters commercializes one such detergent.
It depends how deep into the proteome you want to go. If you are aiming for the top 2000–3000 proteins , then no offline fractionation is necessary. For higher protein IDs, minimum of 6 fraction with SCX or pH 10 separation going to increase your protein ID by 2-3 fold compared to no offline fractionation. Good luck
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