New SP3 protocol by Hughes et. al. that might be as good as FASP, but easier.

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New SP3 protocol by Hughes et. al. that might be as good as FASP, but easier.

Postby MSENC » Tue Jan 13, 2015 4:04 pm

Hi, saw this exciting paper in MSB last month. It maybe as good as FASP without some of its shortcoming. The magics seem to be the magnetic beads with carboxylate surface. Anyone has any more insight on this protocol?

The single shot data is very impressive in term of the annotation rate and total protein IDs, when I downloaded the data and put them into our own pipeline.



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Postby JDRCHEM » Wed Jan 14, 2015 8:03 am

I am curious how the resin they used is able to bind more than 100 times its weight in protein. They claim that 1 ug of bead can bind 100 ug of protein. I have been doing solid-phase extraction/affinity purification for a long time and I have never heard of any resin having this type of capacity, especially a non-porous magnetic bead, so this would be a first for me. I looked up some binding specs for the Sera-Mag SpeedBeads from GE Healthcare (they bought the mag bead business from Thermo after the Life Tech acquisition) and I couldn't find anything listed for binding capacity for the carboxylate-modified resin, but I found something for their protein A/G resin made with the same mag bead structure. This resin can bind 55-85 ug of IgG with 1 mg of bead. This is totally reasonable for a non-porous magnetic bead and I would expect similar capacities for other coated beads made from the same core particle.

Having only skimmed the manuscript, perhaps I missed something...can anyone explain to me how they achieved this sort of binding capacity with these mag beads? Is there some chemistry that explains this capacity?

Edit: This seems like a question Andy Alpert could answer.

Edit #2: I love the transparent review process. A reviewer had the same question I had about capacity. Here is the question and response taken from the published reviewer comments. This is from reviewer #3:

4. Can the authors provide data on sample binding capacity (protein and peptide) on magnetic beads? Different peptide/protein loadings should be provided to clearly illustrate what is the maximum binding capacity (preferable in the presence of different detergents to illustrate their effect). I'm surprised to see that the HILIC media has a binding capacity of 100 ug of protein per 1 ug of beads knowing that C18 media typically has a binding capacity of approximately 5% on a mass basis.

Author response
e: Data related to binding capacity is present in Figure E3a. The details of the experiment are discussed in the Expanded view methods section (Pg. 6 Para. 2). While we agree with the reviewer that capacity differences in the presence of different detergents is of interest, these details are beyond the scope of this manuscript and are better suited to a more focused audience and journal.


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Postby Christopher » Fri Jan 16, 2015 2:48 pm


To determine the effective binding in the paper, they used differing amounts of BSA to see when the beads started to saturate, and recovered unbound BSA beyond what could be bound. This is by no means a perfect way to do this. What was observed was that around 100ug of BSA could be recovered using 1ug of beads.

This result is a bit surprising, and is likely benefitted by the fact that the beads are 1um, but this still doesnt explain the high capacity. It is more likely that this is due to secondary interactions. As part of the SP3 reaction, a certain amount of aggregation occurs between beads and proteins, but also between the proteins themselves. Due to this, the beads are able to pull down more than their effective surface area due to not requiring a direct interaction with the bead surface.

In practice, and in the protocols provided in the manuscript, they operate a bead to protein ratios far below this determined value.

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