Biomolecule method development issue

[vijayak]

Hi all,
Question 1: I am working on a molecule called recombinant trypsin molecular wt of about 25KDa. At persent i made a method using SEC column but due to some requirements the purity of the same product neded to be establised by reverse phase chromatography. So, as a starting point i had tried C4 & C18(300A) columns respectively using ACN and water 0.1% TFA with a linear gradient. Surprisingly i got a mountain like peak. The moment i tried to break the mountain using some gradient the mountain like peak completely detoriating. Does anyone faced such kind of problems? kindly give me the suggestion.

Question 2: When i inject my compund in SEC(it showes a single peak) and collect the fraction to do Maldi-Tof it showed multiple species ranging from 25-to 29KDA, In such cases can the purity be declared by SEC eventhough the single peak in UV showed multiple species in maldi.

Experts opnion expected

Thanks & regards
Vijayak

[tom jupille]

SEC is not a high-resolution technique, so one possibility is that your protein is, in fact, impure.

That said, reversed-phase is inherently a denaturing technique; it's not unusual to see a "blob" of partially denatured species of the same primary sequence, with each conformer interacting slightly differently with the column. The general approach to the problem is to denature the living daylights out of the protein during the workup (high temperature, acetonitrile, TFA, surfactant) and then to maintain strongly denaturing conditions during the chromatography.

If you're new to protein chromatography, a good place to start is with the book "Basic HPLC and CE of
Biomolecules" by Cunico, Gooding, and Wehr. Here's a link to it on Amazon:
http://tinyurl.com/4vzwscb

[dorota]

I agree with Tom.
You can find many analytical certificates for recombinant proteins with purity determined by rough methods (SDS-PAGE, SEC) but if you really want to know purity of your protein you should develop a high resolution method. To study purity of recombinant proteins I use at least 3 different chromatographic methods.

[pph31]

I agree with Tom.
You can find many analytical certificates for recombinant proteins with purity determined by rough methods (SDS-PAGE, SEC) but if you really want to know purity of your protein you should develop a high resolution method. To study purity of recombinant proteins I use at least 3 different chromatographic methods.

can you elaborate the three methods? are these methods different modes of HPLC such as RPLC, IEX, SEC, et cl?

[pph31]

Hi all,
Question 1: I am working on a molecule called recombinant trypsin molecular wt of about 25KDa. At persent i made a method using SEC column but due to some requirements the purity of the same product neded to be establised by reverse phase chromatography. So, as a starting point i had tried C4 & C18(300A) columns respectively using ACN and water 0.1% TFA with a linear gradient. Surprisingly i got a mountain like peak. The moment i tried to break the mountain using some gradient the mountain like peak completely detoriating. Does anyone faced such kind of problems? kindly give me the suggestion.

Question 2: When i inject my compund in SEC(it showes a single peak) and collect the fraction to do Maldi-Tof it showed multiple species ranging from 25-to 29KDA, In such cases can the purity be declared by SEC eventhough the single peak in UV showed multiple species in maldi.

Experts opnion expected

Thanks & regards
Vijayak

I guess lots of time the monomer peak in the SEC is not pure due to the presence of charged isoforms, major fragments with similar hydrophobicity.

[tom jupille]

If SEC is done correctly, hydrophobicity should not be an issue; the separation mechanism is based strictly on size. More relevant is the fact that SEC cannot completely separate molecules with only a 20% difference in MW. If you have a hodge-podge (I'd like to see *that* word in a chromatography glossary!) of isoforms and conformers, what you will see is a "mountain peak" or "blob" representing the envelope of all the partially-separated forms.

As I said earlier, SEC is *not* a high-resolution technique. It typically has a peak capacity (just what is sounds like: the number peaks you can baseline resolve in a single chromatogram) of 6-10. A full-range reversed-phase gradient will have a peak capacity of 100-200. HIC (hydrophobic interaction chromatography) and IEX (ion exchange) are a bit less. Reversed-phase of course is no good as a prep technique because it will denature the protein. HIC and IEX can both be done under non-denaturing conditions.

[HW Mueller]

Having no experience with renaturing proteins, but seeing a lot of methods that claim that, I wonder whether denaturing during prep reverse phase can be overcome (reversed), generally or in some cases.

[dorota]

can you elaborate the three methods? are these methods different modes of HPLC such as RPLC, IEX, SEC, et cl?

Yes, exactly, one of them is SEC, two of them can be RPLC (with a mobile phase which differ in pH or type of buffer)

I think Vijayak has multiple species (as detected by MALDI) not isoforms and again I agree with the 2nd post of Tom.

[tom jupille]

I wonder whether denaturing during prep reverse phase can be overcome (reversed), generally or in some cases.

I suspect that the reason you see publications on it is that it's exceptional. My understanding is that denaturation is (with rare exceptions) a one-way street.

[pph31]

can you elaborate the three methods? are these methods different modes of HPLC such as RPLC, IEX, SEC, et cl?

Yes, exactly, one of them is SEC, two of them can be RPLC (with a mobile phase which differ in pH or type of buffer)

I think Vijayak has multiple species (as detected by MALDI) not isoforms and again I agree with the 2nd post of Tom.

what would be the typical temperatuer and pore size of your RPLC method?

[vijayak]

Hi tom,
Thanks lot. So what i could understand is that if, i denatuure or reduce the protein with SDS or DTTor guanidine or any other protein reducing agent and inject it will help to get good peak than the 'blob' one.(DID i unerstood correctly?)

Regards
vijayak

[tom jupille]

Yes, you did understand correctly.

Here's an example of the effect, taken from our Advanced HPLC Method Development course. More details are in the reference at the bottom of the figure.

[dorota]

what would be the typical temperatuer and pore size of your RPLC method?

40C, 300A