A very general and perhaps very naive question - does anyone know if there’s been any work toward the equivalent of “PCR” of the proteome?
Instead of doing single worm PCR, can we do single worm proteomics where we multiply the entire proteome of a single animal, and do analysis on that to determine the expression of the whole proteome or of the vast majority of proteins that have been annotated thus far? Also, the input for proteomics studies that I’ve seen reported
in the literature is 240mg of frozen worms or 6mg of protein, does anyone know what the minimum input is? Any references for both worm and/or non-worm models would be appreciated.
To my knowledge, the answer is no. Single-cell transcriptomics and genomics are feasible due to amplification steps, which allow for detection of lowly abundant signal. There are chip-based single-cell proteomic platforms, but these detect only a limited number of proteins. http://www.ncbi.nlm.nih.gov/pubmed/22203961
For the proteome type analyses that you’re discussing, mass spec is required and there is no amplification step currently available. There are a wide-range of platforms with varying detection limits and sensitivities. Access to a good mass spec will really determine how much starting material you need. In your question, is the aim to have a complete proteome for the entire animal? Challenges will be in extracting all proteins from the animal (cuticle proteins in particular need special extraction protocols). Two good recent protocols using SILAC for comparative C. elegans proteomics are:
Thank you. The aim would not be necessarily to extract 100% of all of the proteins, but rather to get at the global changes in protein expression with age or some treatment
at the single animal level or in animals of a particular class/genetic background. I was just curious if anyone is working on amplifying the proteome - that would be a very exciting
addition to the C. elegans molecular tool kit (or any tool kit for that matter!). A 1ug input seems promising. I need to read up a lot more on this, thank you for the references,
they are very helpful.
as Dr. Zimsky in the Film ‘Core’ said in response to a scientist saying “that’s not possible (getting to the centre of the earth)”…“but what if it were?”
There is a targeted approach called Multiple Reaction Monitoring Mass Spectrometry that can be used to quantify changes in many candidate proteins (ok not yet the entire proteome as this would lead to sleepless nights).
If quantitative proteomics (ie. mutant vs WT or comparing aging animal proteomes) is what you’re after, then I would recommend SILAC. The protocols are in the two links that I previously provided. The power of SILAC (stable isotope labeling by/with amino acids in cell culture) is that it allows quantitative proteomic comparison of two conditions. You would need to grow the strains to be compared on heavy and light media . ie. the bacterial strains carry heavy and light lysines isotopes. Then extract the proteins, and analyze equal amounts of input. SILAC has been a really powerful tool in a lot of systems. A similar approach (ITRAQ) allows labeling and comparison of up to six conditions, but I’ve heard from a colleague that the results are quite variable.