Hello,

I am using the phenomenex polymer x column to separate peptide mixture and identify them by LC MS/MS. Since my samples are all trypsin digested and I do have trypsin in these typically I should see the trypsin peak along with the peptide peaks. But I dont see either the trypsin or peptide peaks. I tried infusing a std peptide to see if my tune file works alrite and it does ..so am assuming my MS is working good. Then I made a new column of polymer x but this did not help either. I tried different protein samples and got nothing again. I injected std peptide on my column and get peak for it but with much less sensitivity than direct infusion. But when I spot the sample on a maldi target I get beautiful Maldi spectrum and I can see trypsin peak and lots of peptide peaks. I check the flow rate of the column and its alrite. I made sure there is no clog in the plumbing. Any suggestions will be greatly appreciated.

Thanks!

You really don't have a clue how to do the LC part of this LC MS/MS do you?

Here are some suggestions (as requested). First, start off with a little reading, the lit. is full of good papers and books on the topic, then buy yourself a decent column to do the work.

What is your LC flow rate in comparison with your infusion flow rate? What is your mobile phase?

Are you sure that your autosampler is drawing the correct amount of sample? Also, if your injection volumes are small, you may have a leak or even a clogged injection port and not be able to see wetness in the area. What kind of autosampler are you using? Is there a switch valve on the MS? If not, insert one and try injecting from the switch valve.

the flow rate of my column is about 4uL/min while that of the infusion is 3uL/min. And my mobile phase is ACN and water. I use a gradient starting with high % of water.

OK, those flows are similar enough that the same source conditions should work. A simple experiment to eliminate your column as the cause, is to replace it with a union and do a flow-injection of one of your pure standards. If the flow-injection looks bad, then you have a leak, or your autosampler isn't injecting, or you have too much dead volume.

Oh yeah, and I'm assuming your column and tubing bore is appropriate for your flow rates.

are you using stainless steel tubing, also try to passivate(sp?) your column, it may help.

I dont use an autosampler. I did check the flowrate at the spray needle and its alrite. I checked the sample loop for any clogs and there seem to be none. Also my labmates who analyze small molecules are getting wonderful results using the same instrument but a different column. All this makes me think that there is no clog anywhere. The weird thing is when I infuse 25pg of std peptide I get wonderful peak but when I inject the same std I can see peak only at 5ng on column which is crazy as chromatography has to be more sensitive than directly infusing the sample right? I also packed a new column of the same packing material and that did not help either!! I think the packing material has gone bad but I dont know how to check that

How does a flow-injection (no column) of a standard look?

flow injection of a std peptide looks alrite.

By alrite, do you mean sensitivity is near the expected level? If so, then it sounds like your column is the problem. What are the column dimensions and particle size? What is the width of your peak coming off the column as compared with flow injection?

yes sensitivity is good with flow injection. And am guessing too that my column has gone bad. But I dont know of any means to check that. I pack 15cm column with polymer x from phenomenex the particle size of which is 3um. I use a 440um OD capillary.

oops I forgot to mention the capillary id its 320um

Your flow rate seems reasonable for that dimension of column. You said you can see your compound eluting from your column, but only when you inject a huge amount. In that case, is it a big ugly broad peak, or a nice sharp peak?

Another question to ponder: are you sure your compound is really eluting from your column?

When I directly infuse the std peptide I can see nice peak for 25pg while when I inject it I can see peak only at 5ng on column. The std peptide that I inject is vasopressin and I get nice peak at 1084 and 542 which are the singly and doubly charged vasopressin.

Perhaps I was unclear. Your MS sounds like it is working fine. I was inquiring as to the shape of your chromatographic peak (either in the TIC or extracted ion chromatogram). It should be about as sharp as your flow-injection peak. If it's overly broad, you might have voids in your column. If the peak got smeared out like this, you would lose sensitivity.

Another thing to ponder is whether your compound is really eluting from your column. Or more generally, whether your LC conditions are appropriate for your peptide. For example, is there appropriate pH control of your mobile phase? Is the pH control in your mobile phase helping or hindering MS sensitivity, or are there ion pair reagents in your mobile phase that are hindering sensitivity? Is there enough organic near the end of your gradient to elute the compound?

If all else fails, you could purchase a column from a known good supplier. Then you won't have to worry too much about whether the column is properly packed.

MG thanks for all your help. I finally got to check with the phenomenex people and they said the column could have gone bad. I am going to try to use a different packing material and see if that works for me. Thanks once again.

I don't know about that.
The Phenomenex service people probably could not figure out the problem either, and to get you off their back, they suggested to use a different column...
Come back, if the issue reoccurs!

I tried a C18 column and that does not seem to give me any peptide peaks either!!! The LC method and the tune method that I use worked just fine before so I dont know what is wrong now. The weird thing is sensitivity has decreased with the column. I can detect only upto 5ng of standard peptide on column but when I directly infuse I can go down to 25pg. What does this mean? Does this point at something.

Wait, you just said your LC method worked fine before. So until this recent problem, your peaks eluted from your column and you had adequate sensitivity. Right? And you've tried another column and also get poor sensitivity. This makes me suspect that something is wrong with your LC system. You've tested it with flow-injection and everything seems to be fine. So maybe you have a leak that only manifests itself at high pressure, somewhere upstream from your column. Maybe it is your injection valve leaking, as was suggested by Anon 4/6 7:23am.

But, you do get a little blip of signal from your standard if you inject enough. That is a start. When you inject 5 ng so that you can see some signal, how does retention time and peak shape compare to when the method was working? Are you sure you are using the same conditions that were known to work? Double check your method.

I have checked my LC method and its the same one that I used when I got lovely peaks for my sample. Now that you mention it, the shape of the peak (5ng on column) is not a neat one but slightly distorted. My labmates are using the same instruement but different column and getting nice results (can detect 2pg of their small molecule). So I am guessing there is no leak at the sample injector. I am tuning the LCQ for a std peptide now and lets see how it goes.

Same method, with same column type, on same system gave good results. Now with new column, results are bad, but colleagues have no problems with their methods. Double-check that you really are using the same conditions as before, and check your plumbing. If mobile phases are old, prepare new ones. If mobile phases are new, maybe prepare them again anyway, making sure you are using the same recipe as your original (working) method. At those flow rates, you'll only need small amounts of mobile phase anyway. If everything else checks out, it sounds like the problem there is your column. I'm about out of ideas.

I prepare fresh mobile phase every time I use the instruement. If it was a problem with my column, which is what even I thought, how come I did not get results when I used a column with a different packing material.It looks like I have hit the wall!!

OK, here is a simple issue that got me at some point: I confused the A and the B line on my system. With this, I got only unretained peaks. Of course, I felt really stupid afterwards...

I think I finally solved the mystery. I tuned and calibrated the MS detector with the calibration cocktail. Then tuned it for one std peptide. Now I injected my peptide mixture and I can see peptide peaks. Finally!!!! Thanks for all the helpful discussions.