Dear All,
I am planning to do NanoLC-MS analysis, as I am doing this for the first time and I have no idea about sample preparation. I have gone through some of the article and I found they are using columns for peptide sorting and desalting purposes. But as I do not have those things in my lab so I can not follow their protocol. So, If anyone of you having experience with the same then kindly let me know the protocol that do not needs any special requirement.
Looking forward for your help and suggestion.
Dear Amit
You need to provide a bit of information to go on.
Do you have the equipment to work on or are you submitting the samples to an analytical laboratory that has the equipment. NanoLC is not a trivial technique and it takes quite a lot of patience and experience to get reproducible results using this technique. A few questions:
Are you doing in gel digestion or solution digestion?
Are you using trypsin as the digestion enzyme?
What is the origin of your sample?
How much sample do you have?
Are you comparing different conditions, only looking for protein IDs, doing quantitative analysis using labelled or unlabelled techniques?
If you google "LCMSMS protein protocols" you will find many detailed protocols from both Journals and universities.
Good luck
Dear Duncan,
First of all I am very much thankful to you to show your keen interest for my question.
As you said I can go with Google in search of related article. Actually, I did find a nature protocol for the same but in that they have used some columns for peptide size sorting and digestion and I do not have those things in lab. I am also finding other article but I am getting confuse which I should use.
Here, I am writing a brief answer of question. kindly read it and share your experience like what all precaution I should take and which lab method would be good for my work.
I have NanoLC-MS in our institute's central instrument facility.
Actually, I am trying to look for the abundance of different nucleus proteins in normal and tumour sample. It is a comparative analysis and I will go with the unlabelled quantification of the same(this is also new to me but I am reading related articles). Secondly, I have tried with cell line not with tumour samples, with lysis of 8 million cells I am getting around 300 ug of protein and now I am planing to start with tumour samples (samples are not much so I am not sure how much I going to get) and I would like to go with in-solution digestion.
I would like to share further information (if requires).
Dear Amit
I would isolate the cell nuceli to start with as this removes the more abundant cell proteins that will make quantitation of your nuclear protein fraction difficult.
Check this link. http://www.celldeath.de/apometh/nucprep.html
You can also use a ball-bearing homogeniser with the same buffer.
The use of the protease inhibitors is important so do not omit these, but you can use protease inhibitor cocktail tablets or solutions.
You will need to chech the protein concentration of the isolated nuclei and will need at least 10 ug protein to work with. This must be done accurately for label-free quantitation.
Digestion can be done in solution but this will also need to be very well timed, temperature, protein to enzyme concentrations etc as it will impact on the reproducibility of the method and you are only really going to be able to reliably quantitate large fold changes using label free methods.
Follow the protocols from Nature or Springer Protocols as they are well tried methods that work.
Good luck
Thank you so much for your valuable suggestion. I will try the well establish protocol. Thanks
Amit
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