PEG, being a polymer, tends to interfere with matrix crystallization. As a result of this, it is difficult to produce decent signals in MALDI-TOF (linear positive mode).
Would you have any advice (or protocol) that could share for MALDI-TOF analysis on PEGylated large proteins (e.g. > 100 KDa)?
Apart from MALDI-TOF (or mass spectrometry), Are they any alternatives to characterize PEGylation on proteins? Thanks.
what matrix u used? You can try to use different / combined matrix.
Hi Kay,
for my research I often deal with PEG-Peptide conjugates (up to 50 kDa, but not higher). The matrix I use is HCCA (α-Cyano-4-hydroxycinnamic acid) for all conjugates. I have it as a saturated solution in TA-30 (30% Acetonitrile in water with 0.1 % TFA).
First, I apply 1 µl of that solution on the target and let it air dry. I do that freshly for every sample spot.
For Analysis, I use an analyte concentration of 0.1 mg/ml in TA-30. I mix that 1:1 with saturated HCCA in TA-30 and apply 1µl on the 1µl dried matrix spot (dried droplet method). With this, I get good crystalinity.
Also try to use fresh saturated matrix solution, for my experience, this has the best crystal "behaviour".
Play around with concentration and maybe salts (e.g. NaTFA). Do not use AgTFA, this precipitates with cystein-containing samples. I would not recommend to mix matrices for the first try, then you'll have too much parameters (and solvents) which you can not overlook.
Maybe you could search for an application at the Bruker website. Some years ago I was at talk of Bruker about Top-down analysis by in-source dissociation. I think they showed some data about disulfide bonds and pegylation. Maybe this publication might be helpful:
http://www.sciencedirect.com/science/article/pii/S1044030508008714
If you reduce and alkylate your protein you could try to do top down sequencing. When sequencing stops at the position of a lysine, it's most likely that there is a pegylation site. However, I don't know whether your MALDI is suitable for ISD and whether you have a software to analyze the ISD data. Additionally, you also might have multiple PEGylation sites. To test this, I would recommend to acquire spectra in normal mode. If you know the size of Pegylation (i.e. average PEG5000), you can easily go to the peak of the PEG-Protein mountain and calculate the number of PEG chains attached to the protein.
Hi Kay,
good luck with your research, too. You can let me know if you have any further questions.
I often use the (i would call it) "double Matrix" method which I described above. If I compare the same sample, I often have less noise and better signal intensities with the same laser power for the "pre dried" matrix spot plus sample in matrix solution in comparison to the method without pre-dried matrix spot.
Marcus
Hi Wong,
Pegylated proteins are difficult to deal with and give two problems one as you mentioned about co-crystallisation with matrix, second the differential ionization.
To prevent the matrix problem, what i suggest is to use lawn preparation, basically you mix CHCA in Acetone saturated solution and then layer it as thin film on target plate, after drying, add one ul of your sample protein on top and allow it to dry.while shooting the laser, concentrate on the rim of the droplet, thats where most energy transfer will take place. You can try some water soluble matrix also like DHB.
If you have the option of ESI-TOF, you can use a charge-striping agent like TCEP and add it post column to suppress multiple ionization of PEG envelop and you will get a complex spectrum which you can deconvolute.
I have seen both approaches work for PEG-protein upto 40 kDa but nothing beyond that.
Hope it helps.
regards
Saurabh
Hi Saurabh,
May I ask a follow-up question regarding the 'CHCA in acetone' matrix solution?
Did you use 100% Acetone in a saturated solution of CHCA? But in many cases, this matrix would spread considerably on the MALDI plate.
or May I know what percentage of CHCA matrix / acetone did you used? Thanks again!
Hi Kay,
I hope I can give a small clue. Aceton due to its low surface tension spreads well over stainless steel targets (I guess thats what you use?). First thing, kind of trivial: Apply a lower volume on the plate (e.g. for TA-30 I do 1µl; if I have THF as a solvent, it's only 0.3 µl).
From e.g. Bruker, there are many different target types available: "normal" stainless steel, polished steel (where the solvent spreads even more), or anchor chip targets (here, the stainless steel target is coated with a hydrophobic polymer, only on the target spots, there is no polymer. In this case, the solvent stays in place without spreading too much). I guess, the anchor chip type would be good for you in that case. This type of target is available with a prespotted HCCA matrix as well.
I attached some links for you with description (I think the last link is the best, because it has the most measurement examples). You need to copy the 3rd and 4th link directly in a new tab, RG does have a problem and does not reconnect you directly... sorry.
At the end, I guess the bigger effect on the spectrum is gained with the right matrix/analyte ratio, concentrations and most importantly - the right matrix, of course.
http://www.bruker.com/products/mass-spectrometry-and-separations/literature/literature-room/instruction-for-use.html?eID=dam_frontend_push&docID=54045
http://www.bruker.com/products/mass-spectrometry-and-separations/literature/literature-room/instruction-for-use.html?eID=dam_frontend_push&docID=49082
http://www.ispybio.com/search/protocols/maldi protocol1.pdf
http://www.rib.okayama-u.ac.jp/saka/MALDI-TOF/Images/AnchorChip Manual_2_0-1.pdf
Hi Kay
Things to consider are the sizes of protein and PEG; mono versus multi-PEGylated; whether analyzing a reaction mixture or purified PEGylated protein; presence or absence of unconjugated components (PEG or protein);
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