Hi everyone,
I come across some information and publications online that recommend sample desalting after sample preparation (denaturation, reduction, cysteine blocking, digestion) prior to fractionation with strong cation exchange (SCX) HPLC column. I am working on 8-plex iTRAQ proteomics protocol and the manufacturer's guideline only highlighted desalting step prior to RPLC-MS/MS so to prevent blockage of the nanoHPLC column.
As far as I know, the buffers meant for SCX fractionation contain high concentration of salt (especially 1M KCl). So, is it necessary to desalt the peptides before fractionation? Some publication did not mention about doing so, which I assume they just load the peptides in for fractionation as it is without additional clean up step.
Can anyone give me some advice and opinion on this issue?
Thanks in advance.
H. K.
When a cation exchanger is use, the sample (peptide/ protein) is loaded at a slightly acidic pH and the buffer shall have a low ionic strength (Say 20 mM Phosphate buffer). Thus it is necessary to desalt the peptide for its optimal adsorption/ binding to the cation exchange matrix.
When a cation exchanger is use, the sample (peptide/ protein) is loaded at a slightly acidic pH and the buffer shall have a low ionic strength (Say 20 mM Phosphate buffer). Thus it is necessary to desalt the peptide for its optimal adsorption/ binding to the cation exchange matrix.
Partial desalting must be performed before IEX and total desalting before HPLC-MS.
Hi Hong,
If you are doing the 8-plex iTRAQ that's mean you have a volume of around 800 ul and in here you have urea and other salts. I will recommend you to desalt and dry the sample before the SCX fractionation, you will get rid of the salts and you can reconstitute the label-peptides in an adequate volume to load into your SCX column.
In some cases I used the iTRAQ labelling, but I don't do the digestion as they mention, I used my own way of digestion, then I do clean the tryptic peptides before the labelling (labelling is done as recommended by the supplier). In this case I only mix the iTRAQ flavour and dry this, then reconstitute this into the appropriate buffer for SCX fractionation.
I hope this is helpful and good luck
Hi Shamsher S. Kanwar and Omar Gonzalez-Ortega ,
Thank you for your advice. I will take note on that. I wasn't able to find any explanation on this issue anywhere else and we don't have any expert in our laboratory. So, your advice is very valuable to me.
In addition to that, is it possible to use Pierce C18 spin column to clean up and remove Tris buffer from protein sample before denaturation, reduction, cysteine blocking, digestion and labeling? We use RIPA buffer (readily available in our lab) for extraction and I am aware it contains Tris buffer that could react with iTRAQ. I have tried acetone precipitation and methanol precipitation. Both result in pellet that is impossible to reconstitute. Unfortunately, we don't have pulse sonicator to assist in the process. So, I am wondering whether Pierce C18 spin column can help with removing Tris.
Hi Martin E Barrios-Llerena ,
Thank you for describing your work with iTRAQ. Yes, your input helps me out a lot. We have speed vac and freeze dryer here. So, my initial plan is to dry the sample by speed vac after labeling with iTRAQ to reduce the volume because we only have analytical HPLC here for me to do fractionation. I intend to reconstitute the labelled peptide to around 40uL which I can inject twice for fractionation (max load is 20uL for this HPLC and SCX column capacity is 38mg/mL). This part is where I find some recommend desalt while some did not mention at all, which I presumed they did not desalt before IEX. After fractionation and desalting with Pierce C18 spin column, I am still considering whether I should dry the peptides by speed vac or by freeze dryer. We are going to send the sample to another university to run ESI-MS/MS.
As for digestion, I am using Promega Tryp/ Lys-C. So, I am going to follow Promega's protocol to digest overnight.
I really appreciate all the input you and the rest could offer because I can only have one go with iTRAQ. The kit and ESI-MS/MS service is costly here. So, I must nail it in one go. Currently, I am planning a mock run without labeling to see how many peptide fractions I need to process and to optimize with ESI-MS/MS service provider before the actual run.
Lastly, my another concern is the Tris buffer which comes with the RIPA buffer that we have. As per my response to Shamsher and Omar, I am also considering the possibility of using C18 spin column to clean up protein before I start preparing the sample for digestion and iTRAQ labelling.
Thank you again for everyone's response.
Hi Hong,
I will try to explain how I do my iTRAQ experiment, and you can take whatever you feel is convenient for you.
First for the protein extraction, do you cells harvesting as usual (washes with PBS and sos on) and then for the protein extraction (if you are interested mainly in cytosolic proteins) prepare a buffer containing 8M urea in 50mM HEPES pH 7.5. Use some means of cell disruption, e.g. sonication probe, sonication bath, glass beads and vortexing, etc. Centrifuge at max speed in a bench top centrifuge for 15min at 4 celsius. Collect your supernatant, do a protein quantitation, and this solution is ready for digestion with Trypsin.
After digestion clean with a C18 cartridge for the adequate amount of protein. SpeedVac the sample, dried sample is ready for iTRAQ labelling, dissolve the peptides in the buffer recommended by Sciex and label accordingly to the protocol provided. Combined the iTRAQ flavours and SpeedVac.
Now to do the SCX, if you have a PolySULFOETHYL A SCX column of 2.1x200 mm you can easily load the whole iTRAQ 8-plex, or divide the sample and inject half.
Collect your fraction, desalt and is ready for MS.
I hope that this help you, good luck again
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