Natural ligand lost after HPLC

[pipettemonkey]

I'm trying to purify a natural peptide ligand from brain tissue. This can be challenging because I am starting with, at most, 10 pmol. Someone (Uwe?) used the analogy of finding the needle in the hay stack. That is exactly what it is like.

After 5 or so HPLC steps, the purification was going well; much of the protein/peptide contaminants removed. However, when I got down to the 2.1 mm ID sized cyano column, I lost my sample following HPLC (I could no longer detect it). I have a sensitive assay that requires me to burn < 1% for analysis, but I analyzed up to 10% of each fraction and still saw no activity (i.e., it is gone.. or, the machine "ate it.")

I was able to use the same cyano column with success a few days before to purify a different (but similar) natural ligand from the same brain tissue (actually, both ligands were put through the exact sequence of steps). However, when I took the active fractions from this step, diluted with 2-3 volumes water, and injected them on a 1 mm C8 column. Once again, I lost my activity, but on a different column.

This makes two columns that I have lost activity on. Yet, I have used both of these successfully in the past to purify natural ligands, so I'm not convinced it is the column. In fact, this is the first time EVER in ANY HPLC step that I have lost my activity.

There are two differences between this purification and ones in the past:

*I started storing my fractions with 0.% HFIP (to prevent adhesion to the PP tube during storage). (I started this at the beginning, when everything was working fine). I have always kept the fractions at 4C until the next step. I never dry anything down before re-injecting; I just dilute with 2-3 volumes of water.

*I am using a different sample loop. I had been using a 2 mL stainless steel loop. However, in order to inject larger volumes, I borrowed a used 5 mL stainless-steal Rheodyne loop from a neighboring machine.

All else, including instrument, columns, mobile phase, solvent program, etc, is the same. (I -did- change the pre-column frit at the beginning of the purification. )

Could it be something with this loop? (I DO make sure the loop is emptied before re-injecting sample/starting run). I'm thinking, perhaps, it is corroded, and may trap the ligand now that my peptide is relatively pure (and ultra low-abundance). Perhaps this was not of consequence in earlier steps because the impurities were of high enough abundance to prevent such occurrence.

One suggestion I got was to passivate the system with 6 M HNO3 and to flush the columns with EDTA over-night to remove traces of metal ions. I will do this over the weekend. Does anyone have additional advice?

[HW Mueller]

Is this a stable isotope detection?

[pipettemonkey]

This is a cell-based reporter assay. No chance of the assay not working.

[HW Mueller]

Thought you are literally burning analyte.

What is a cell based reporter assay? If it is a bioassay which requires a prior chromatography due to low specificity I wouldn´t bet one cent on it inj your case.

[pipettemonkey]

Thought you are literally burning analyte.

What is a cell based reporter assay? If it is a bioassay which requires a prior chromatography due to low specificity I wouldn´t bet one cent on it inj your case.

The assay is really straight forward and reliable:

I dose receptor-transfected cells with aliquots from each fraction, then measure receptor activation using a cis-reporter. I've isolated a separate ligand using this approach. It's not as sensitive as ELISA, which is the better option if some sequence information for the ligand is known.

[HW Mueller]

We did something like that with ELISA (for those who are not familiar with this: these are immunoassays) detection. It turned out to be extremely sensitive to organics in the sample solvent, among other things.
How do you know that you didn´t have any problems before? The only way that I can think of that allows one to notice there is a problem is when you don´t see anything when you should. In any other case you always get a number (result) with bioassays, they don´t give any hint on partial malfunction.
Stated differently: I don´t think that one can use a bioassay to quantitatively check a chromatographic system.

[lmh]

Yup, bioassays have their problems, but there are things you can do to check for this. Do a blank run of the suspect method where the ligand disappears. Mix fractions of it with an extract that definitely contains the ligand, and try the bioassay on these fractions. If there is no activity, then something about the fractions has nobbled the bioassay. If there is activity, then you should have detected anything that came off the column. Note that this can be done quantitatively: if there is bioassay, but it's only 10% of what ought to happen given the dilution of active fraction by suspect-run-fraction, then there is still something disturbing the bioassay.

[pipettemonkey]

We did something like that with ELISA (for those who are not familiar with this: these are immunoassays) detection. It turned out to be extremely sensitive to organics in the sample solvent, among other things.
How do you know that you didn´t have any problems before? The only way that I can think of that allows one to notice there is a problem is when you don´t see anything when you should. In any other case you always get a number (result) with bioassays, they don´t give any hint on partial malfunction.
Stated differently: I don´t think that one can use a bioassay to quantitatively check a chromatographic system.

At least until I get down to the 1 mm ID column size, I can *see* a peak for the ligand. Sometimes the ligand is of such low abundance that I don't even see a peak using a 1 mm column. I use the assay to determine the active fraction(s).

I assay an aliquot from each fraction. From the signal generated in the bio-assay (compared to a positive control of crude extract), I try to gain an idea of how much I recover from each step.

[HW Mueller]

What if your "crude extract" gives cross reaction?

Did you do a study showing that all solutions coming from your different chromatographies are compatible with your bioassay? Even if it seems to work I would expect this to be semiquantitative at best. Also, I wonder why you take small aliquots if there are situations were you are slipping below what you consider the detection limit?

[pipettemonkey]

What if your "crude extract" gives cross reaction?

Did you do a study showing that all solutions coming from your different chromatographies are compatible with your bioassay? Even if it seems to work I would expect this to be semiquantitative at best. Also, I wonder why you take small aliquots if there are situations were you are slipping below what you consider the detection limit?

I dry the aliquot before assay. I normally use TFA, CH3CN, or sometimes 1-propanol, or otherwise volatile solvents in the mobile phase. I've used all these solvents in past purifications with no problems.

The reason I take small aliquots is so I don't spend too much of the sample after each step (expect 6-8 HPLC steps to purify to homogeneity). If I had to use, say, 5% just for analysis at each step, I wouldn't have much left after the last step.

A small (<1%) aliquot typically gives me 2,000 % of vehicle treated. I upped the aliquot to 10% and saw nothing in the last two runs where I appeared to lose my ligand.

[HW Mueller]

Whew, you are optimistic, doing a purifiction sequence without knowing that your individual chromatography or detection steps work.
If a sequence really worked with one peptide (in my pessimistic thinking I wonder whether you really proved that) it does´t necessarily work with another.
OK, you seem to have eliminated possible organic solvent interference, but what about salts, etc.?
Another story, I think this was mentioned at least partially before, here: I was involved in isolating ouabain (a steroid) from a human body fluid and got nothing. I did notice, though, that ouabain has a fairly good tendency to carry over due to Rheodyne injectors. I asked one of the persons from another lab which had support for the hypothesis that ouabain is a human hormone. I found out that they never checked into the possibility of carryover. To shorten the story: The "noise" created about ouabain being a new human hormone has quieted down quite a bit. I am convinced that it was found in body fluids via carryover from standards, it doesn´t seem to exist endogeneously in us.