Dear colleagues,
Hi. I am new to LC-MS/MS proteomics and would like to seek you advice on a few LC configuration:
Thank you very much, appreciated!
Hi,
Even though it depends on the type of nanoLC system you're running, trapping of peptides on a pre-column not only allows you to clean the sample from salts and other interfering compounds before analysis, in some cases (where the trap column can be switched from the system) it will also enable sample loading at relatively high flow rates, which saves analysis time. So there are definitely advantages to using a trap column.
It's quite obvious when a trap column needs to be replaced (most often these are not cleaned, but simply replaced by a new trap column): if the pressure during sample loading increases dramatically, this means that the trap column is blocked/dirty.
If you're envisioning to run on-line 2D-LC, you're most likely running SCX-reversed phase? In that case, trapping of peptides eluing from your SCX column is required to desalt prior to the second dimension (reversed phase) separation that is coupled to your MS. Trapping prior to the first dimension (SCX) is not routinely done, I believe.
Hope this helps, good luck!
Dear Kay,
The trap column serves two functions: to concentrate and to clean up a peptide mixture ready for LC-MS/MS:
1. If you have a large volume of sample, you can use the trap column step to load all the material at a fast flow rate eg., 20 microliters/min before isolating the sample loading pump and placing the trap column to be on-line with the nano-LC MS system. So the trap column here serves as a step to inject large sample volumes in a short time, on the LC-MS/MS column
2. Trapping step also serves to clean some of the interfering compounds that may be present in the peptide sample, before the mixture goes on the analytical column.
I always carry out peptide clean up/desalting step before LC-MS/MS. So, in my case, the trap column serves as a way of concentrating and introducing the peptides in a volume of ~5-10 uL of 0.1% formic acid onto the trap first, then to the analytical column.
The nano-LC MS systems usually run at 200-300 nL per minute flows. So if you do not have a trap, it will take you a very long time to inject a sample. Trap or loading pump is running at uL/min, and it is an easy way to overcome the issue with how to inject large sample volumes on nano_LC systems.
For a 2D nanoLC system, you flush the loading side with 0.1% formic acid, eg., 20 uL /min until a steady pressure reading is seen. The analytical column side flows at a much lower flow, eg., 200-300 nL/min so depending on the inner diameter and length of your analytical column, the equilibration with 0.1% formic acid may take 20-30 min.
You may need to optimize the time it takes for the sample to be pumped from the autosampler to the analytical column. You can do this by comparing the data obtained on a direct injection experiment (eg., 1 ul of the sample injected directly onto the analytical column) vs, an injection done with on-line trap loading system.
3. It is good practice to keep a log of the pressure of the trap column and analytical column at the operating flows. When you see a pressure spike during trap column equilibration, (and the pressure stays high), then it is time to change your trap column.
All the best,
Hediye
Dear Kay,
I completely agree with Eef and Hediye. Apart from that, It is definitely advantageous if you use a trap column in 2DNano-LC setup as that will increase the number of proteins that could be identified using the MS system. Trap columns are relatively robust as compared to the 1D or 2D Analytical column, having said that they can bind more peptide sample (concentration) as well as they can resist more flow rates. If you are planning to run step gradients, trap columns are a must as you can increase the number of steps (concentration of salt) to elute the peptides.
If you are planning for SCX-RP or WAX-RP, you may consider using volatile aqueous buffer systems such as Ammonium formate, Ammonium acetate etc...
All the best for your efforts
Saravanan K
To the already mentioned points I just want to add the fact that the pre-column concentrate the sample from a large volume to a small volume and therefore the individual peptidespecies are eluted very sharp at the following LC-MS run, you will get much smaler peaks for each petidspecies . Otherwise, a peptide at in a 5μl sample volume and a running speed of 200nL/min will elute in a peak of just over 25 minutes ... Therefore you'll not expect a high signal for this peptide. But with the pre-column the same peptide will elute in a range of perhaps 1 minute!
One additional point is that the practice of using pre-colum is a good one since it extends the life of the, usually more expensive, analytical column. Your analytical columns last a lot longer when you have a pre-column that takes care of any salt or particulates that have made their way to the nano-LC system.
Best, Hediye.
Dear Kay,
In addition to the above valuable advices, a good practice is to uncouple the trap from analytical-column before reach high concentrations of organic in the eluent (60-70% ACN is enough to elute peptides), to prevent that the highly hydrophobic compounds may reach anyway the analytical column and clog it. When the trap is uncouple wash it thoroughly with 100% organic.
All the best,
Francesco
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