The total concentration of peptides in the solution might be very low, less than 100ug/ml. I am not sure how many kinds of peptide are in the solution. I plan to identify them by MASS. I also plan to remove the sugar by ethanol and phenolics by PVPP in the solution before peptide isolation. I am not sure whether it is necessary or not and if I should worry about the ethanol and PVPP might remove some peptides as well.
You can try C18 solid phase extraction from acidic solution (5-10% acetic acid). Sugars will not retain unless they are chemically and extenssively modified. Small phenolic compounds will not retain either. Wash C18 SPE cartridge with 0.5% acetic acid in water and elute the peptides with 80% acetonitrile 0.5% acetic acid. Dry the solution in vacuum.
Dear Nikolay, thanks for your answer. It is very helpful. Could you provide more details about the C18 spe cartridge, such as from which company I can buy. Sometime it is really difficult to find some special chemicals for research in China. I had though about the c18 ZIPTIP from millipore but it looks so small. I am interested in the peptides with MW < 10,000 Da. Could you suggest which MS mehtod I can use for the identification, such as which kind of ionization mehtod, Maldi or ESI, tof/tof or others? I only used maldi tof/tof for my protein identification.
Hello,
PVPP is good for phenolics removal, not sure how it might remove some of the peptides.
C18, is a good approach, ZIPtip might be small, but the 10ul tips can retain up to 5ug of peptides that is a lot for mass spectrometry analysis, and is the right amount for nanoLC-ESI MSMS. Ziptip and other tips in 100ul version exist. You will find C18 material from most company. I think Waters and Thermofisher have device for ml range sample SPE with C18. I kept to the small format tips, but because my samples are rather diluted, as most plant protein samples are, I load the tips several time, estimating that I am loading 2x to 10x the protein content amount. However I would first deplete the sample of larger proteins as described below.
I had a difficult plant/fungus sample with little biomass and proteins, and wanted to enrich the extract for smaller proteins/peptides by differential Acetonitrile precipitation, or run my protein extracts on 12-15% Tricine gels followed by in gel digestion prior nLC-ESI MSMS. Such gels are good to separate small proteins and peptides below the 10KD range and a way to purify samples from sugars (1-20KD is well resolved, see the nature method paper by Schagger on tricine gels). You can also check my publications where I discribe the workflow for these in Bindschedler et al (Methods, 2011) and maybe also sup data of Spanu et al (Science, 2010)
Hope this will help
Laurence
For 10 -15 ug of peptides you can pepare and use a C-18 stage tip - Juri Rappsilber, Matthias Mann & Yasushi Ishihama; Protocol for micro-purification, enrichment,
pre-fractionation and storage of peptides for proteomics using StageTips, Nature Methods, 2(8), 2007, pp. 1896-1906.
Run a 2D gel electrophoresis then just take a pipett tip and "pick" the spot you want to identify (assuming you know the isoelectric point & mw of the protein you want to separate). It worked for my lab :)
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