Here's the replies to my IMAC query that were not sent to the list.
Identifiers have been removed.
I am happy to see your question. My experience with IMAC zip tips has been
exactly that. We have not been happy at all with recovery and we have yet
to see any "real-life" sample produce data when these tips were applied. I
quit trying, but I honestly felt that I must be missing something. ???
Please share what info you may get. I suspect that others have had the same
experience.
We have had better luck with just plain old LC-MS/MS using very miniaturized
LC and analysis with our LCQ, but this doesn't cut it when stoichiometry of
phosphorylation is low, as is usually the case.
I usually consider phosphoprotein samples as "mystery samples" because many
biologists who bring me samples have no clue about stoichiometry and they
often have unreal expectations about phosphopeptide analysis.
I think I must have 1000 grey hairs now attributable to "Mystery
Phosphopeptide samples"!!!
There was a article in Anal Chem (1999, 71. 2883-2892, Posewitz, Tempst) on
use of Ga(III).
We have done a lot of work with IMAC and our experience is that it
doesn't work well enough to be of much practical use. Low level samples
seldom work-can't recover anything- and when things do stick and elute,
there is much more there than phosphopeptides. It looks to us like peptides
with high density of acidic amino acids also stick well. We have talked to
people at Monsanto (now Pharmacia) and they relate the same experience. In
short, we have given up on IMAC.
I thought you might like to know that this was a point of
considerable discussion at the most recent Lorne conference on
protein structure and function. The consensus is that IMAC either
works or it doesn't and there seems to be little middle ground. Some
people reported success by trying different metal ions as you
inidcated. It seems to be a lottery.
I guess I'm very down on IMAC as a purification method
for phosphopeptides in general. I think the ziptip IMACs work about the
same as others. I was wondering if you had success using a mini-column
filled with X IMAC resin and Y metal that worked and didn't work using the
Ziptip. Probably especially Qiagen material since it's a different resin
than the rest.
Before I used ziptips I loaded up pieces of plastic HPLC tubing with
Upchurch connectors as frits and IMAC resin. (mainly PerSeptive stuff). I
blindly bound Fe, or Cu to these columns. I say blindly as I couldn't see
that I could never remove Fe from the resin. Haven't you noticed this with
your IMAC tips? Even with a heavy duty EDTA wash the tips still stay
slightly yellow. I think that some of these peptides just stay hooked to
these metals, especially Fe and never come off the column. That's why
Millipore used the different metals to find all of the
phosphopeptides of alpha and beta casein. I'll never trust another paper
that uses these as their test peptides unless they have another dozen that
also proves their case. If you take a close look at the sequence you'll
notice they're chock loaded with Aspartic acids. D, E, H, as expected also
bind. I've used a lot of pH's just trying to bind and elute test peptides
to these columns. If you have to hope your peptides will elute then I guess
I'd use gallium nitrate to coat the column. Or I might start with a Cu tip
and take the "extracted material" and run it thru a Ga tip. Not exactly
high thru-put. And some of the test peptides still didn't bind to Ga. I
won't call it science yet but I think that pSers that are close to Cysteines
never bind to the tips. They are too low to really see in the digest but I
do see them running HPLC & MALDI. Fortunately I have nmoles to run. Other
digests have pulled down some of the pPeptides I found via HPLC but missed
others. In some cases I did see the peptides in the flow thru so they
probably weren't peptides that were permanently stuck on the column. How
can you trust a technique that only gets it right half the time and then
sometimes binds your PO4 so tightly that you can't elute it? Unless you can
account for 100% of your molecule and we know that's impossible out of a
gel, how do you know you have the right PO4 or all of the PO4's that are
"activated"? Is the answer for gels to digest the band with trypsin/Lys-C
and then take 1/2 of it and digest it with pepsin. Take the pepsin half and
run that over a Fe IMAC. I've always had every PO4 peptide bind to it just
not elute. Just hope pepsin did it's job and gave you 5-6 mers that
hopefully will come off of the column.
Elution techniques. Although I was using MALDI I wanted a technique that
would be ES friendly as if I ever worked with unique proteins (ie unknown
gel bands)I wanted something that could work directly. I used 2% NH4)OH
(6.68 ml/100ml). Kept it in a glass bottle but still replaced it every
three weeks. I also tried following with 60% ACN 2% NH4OH to remove
stubborn peptides. Then follow with time consuming 250 mM EDTA. This had
to be run thru a C-18 zip before running even on MALDI. If you find
ANYTHING that completely removes Fe please let me know. I figure if I can
get Fe off the column I should be able to recover my PO4's.
-- ********************************************************************* Dr Len C. Packman Assistant Director of Research Protein and Nucleic Acid Chemistry Facility Department of Biochemistry University of Cambridge 80 Tennis Court Road Old Addenbrookes Site Cambridge, CB2 1GA, UK Tel: +44 (1223) 333639 FAX: +44 (1223) 766002 e-mail: lcp2@mole.bio.cam.ac.uk Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
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