Post in What are people's favorite phosphopeptide enrichment methods and protocols?

Dear Brian,

Thank you so much for your insights!

In your own experience,  SDB-RPS or Oasis MCX , which one gives better result
for peptides desalting?

Rest of post

Thanks!

Best,

Hua

-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian
Sent: Friday, December 11, 2020 4:58 PM
To: <email obscured>
Subject: [External] Re: [ABRF Discussion Forum] What are people's favorite
phosphopeptide enrichment methods and protocols?

Hi Noah,

I've looked at nanomole digests of BSA using LC/UV so I don't have to worry
about SPE prior to MS detection.  I was using a 2.1mmID column and a PDA
detector.  I can direct inject the digest (like in the old days when Edman was
the go-to sequencing method) and use that as the expected result e.g. what a
chromatogram looks like for 100% recovery of the peptides.  Using either formic
acid or TFA in the SDB-RPS workflow, less than 1% is in the flow through of the
SPE.  And no peptides are in the 2% MeCN wash prior to elution.  The run of the
SPE flow through looks like a blank compared to the direct injection of the
digest.  Recovery in the NH4OH/MeCN elution is 70-80%.  This SPE is using the
Oasis MCX not Empore SDB-RPS, 2 x 500ul pulses to elute, but they (Empore and
Oasis) should be equivalent given similar bed volumes. A more exhaustive test
would be a complex digest.  But BSA is commonly used for such testing purposes.

So there doesn't seem to be any struggle retaining peptides.  The SDB-RPS or
MCX retention is via mixed modes.  Possibly two modes:  Charge based and HILIC
based (during loading and initial high organic washing, then charge and
reversed phase after that when washing at low organic concentration).  Given
the high organic concentration, I don't think reversed phase characteristics of
the support plays any role in retention during loading.  The absence of neutral
detergents in the eluates is evidence of that.  I'm not certain about the role
of charge versus HILIC plays in retention given TFA and FA retain equally well.
Andy Alpert might lean toward saying HILIC is less effective in the presence of
TFA but that TFA would interfere with interaction between -NH3 groups and the
-SO4 on the support, therefore, HILIC is the main retention mechanism during
loading.  Regardless, peptide capture is excellent and exclusion of uncharged
or negatively charged hydrophilic (DNA etc.) and hydrophobic (detergents lipids
etc.) is also good.

I can't bring myself to use just reversed phase SPE any longer.  This mixed
mode is so much better because you don't have to worry about detergents and can
be tweaked for other purposes so that opens a whole world of upstream sample
prep possibilities.  For example, I can digest a sample in RIPA buffer, and
this method cleans it up.

You can layer on to this a differential elution scheme as published here: Kulak
et al., “Minimal, Encapsulated Proteomic-Sample Processing Applied to
Copy-Number Estimation in Eukaryotic Cells.” DOI: 10.1038/nmeth.2834.  They
describe a 3-step fractionation of the SDB-RPS retained peptides using a
combination of salt and organic in increasing amounts with the final elution
with NH4OH/MeCN.  You must get the extensive supplementary info for this
article for the complete methods and data.

To remove the +1 charged junk you mention, would be a matter of finding the
right organic solvent composition and ionic strength to wash before elution.
I've not tried doing this, but if you have reproducible +1 junk to follow then
look at the Kulak paper.  Their first eluate for the three-step method is 100mM
Ammonium Formate, 40% MeCN, 0.5% Formic Acid.  A goodly number of peptides come
off in this.  So, tweaking downward from those conditions might wash away the
+1 junk.

Best Regards,
Brian
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4
<email obscured>>
Sent: Friday, December 11, 2020 3:34 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide
enrichment methods and protocols?

CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email
system. Whether the sender is known or not known, hover over any links before
clicking and use caution opening attachments.

Brian,

Thanks for your input. I never saw that second easyphos paper or ignored it if
I did.

Are there any classes of peptides that SDB-RPS struggles to recover? Can it
replace C18 for all or most peptide sample clean-up? We see a lot of 1+ junk in
IP's and some phosphopeptide enriched samples. I'd love to make it disappear.

Noah
――
View topic
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F46wNdpyhY5fEY81J33plFZ&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C6b1bdeba277e4a3337bd08d89e14227b%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637433156599931485%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=oTXUQtLC1tEthVFdEBwng470In%2FuYrwsoGPDCZK%2BfyE%3D&amp;reserved=0
Leave group <email obscured>?Subject=Unsubscribe

――
View topic http://list.abrf.org/r/topic/70CXWCMcwcZyAArCvaZDPB
Leave group <email obscured>?Subject=Unsubscribe

** This email originated from an EXTERNAL sender  to CHOP.  Proceed with
caution when replying, opening attachments, or clicking links.  Do not disclose
your CHOP credentials, employee information, or protected health information to
a potential hacker**.