Posts in ABRF Discussion Forum

Hello everyone,

I'm posting some ~ brief general information about the progress I've made on
membrane proteomics.  Basically, I began using the method in the Brown et. al.
paper to isolate membrane proteins via Triton X114 cloud point extraction.
I've adapted it for my bottom-up proteomics workflow.  The results are quite
striking.  The identified proteins are predominantly enriched for the Cellular
Compartment > Membrane et. al. GO classifications (using PANTHER).  It would be
interesting to use this extraction method following a good cell surface
biotinylation method (http://dx.doi.org/10.1021/acs.jproteome.0c01049) and see
if it reduces the non-specific binding seen with whole cell extracts applied to
the avidin beads.

I don't make it a practice to post such conversational information to the list.
The other reason for this message is to test the GroupServer system to see if
it is functioning properly.

Best regards,
Brian

Rest of post

________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Hampton, Brian
<email obscured>>
Sent: Friday, March 12, 2021 8:52 AM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] membrane protein enrichment

Wow, the ink is barely dry on that publication.  Thanks Jon.

Best regards,
Brian

________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of J.D.Humphries
<email obscured>>
Sent: Friday, March 12, 2021 4:52 AM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] membrane protein enrichment

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.



Yes. Very interesting thread. Thanks.

I happened to notice this paper in my Google Scholar notifications this morning
and thought it was worth sharing:

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling
https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpubs.acs.org%2Fdoi%2Ffull%2F10.1021%2Facs.analchem.0c04970&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Cd80a9376a86447005f4308d8e55e20b1%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637511539868159772%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=A9Bkt4bDp%2BU5JGr2risWBXjLKHlcet0%2F2RDaf%2Fz1AeI%3D&amp;reserved=0

BW
Jon


――
View topic
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F2WOQIHwGD614MvRP6PLiHE&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Cd80a9376a86447005f4308d8e55e20b1%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637511539868169775%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=G3mgvxND6xX6c3AoAtWQGtx1FPlEbDf1t6hXJXa9mpM%3D&amp;reserved=0
Leave group <email obscured>?Subject=Unsubscribe

Thank you for inviting me but I do not work in a core anymore.
Atieh Jalali

Rest of post




On Wed, Mar 17, 2021 at 8:06 PM Mary.Winn <email obscured>> wrote:

> ― 1 file link ―
>
> The Midwest Association of Core Directors is inviting you to share what it
> is like to work in a core and how you got to a career in cores at our April
> outreach event, Why a Career in Cores: Exploring Career Opportunities in
> Shared Research Resource Facilities.
>
> Directed at community college, undergraduate, and graduate students. The
> purpose is to expose scientists to careers in shared research resources and
> the variety of paths available. Will you or your fellow colleagues
> volunteer to share your stories? Register to volunteer at
> https://bit.ly/3ePEv2Y.
>
> We need you to spread the word too! Students and student advocates
> (professors, high school teachers, mentors, coaches, etc.) are able to
> register at the event website: https://mwacd.abrf.org/career-in-cores/.
> An event flyer is attached. Would you send it to at least five colleagues?
>
> This event is not limited to the Midwest. Students and volunteers from
> across the US, Canada, and beyond are encouraged to participate.
>
> Hope to see you there!
> ― 1 file ―
>
>   □ MWACD - Why a Career in Cores.pdf  (276kb)
>
> http://list.abrf.org/r/file/uy1Fl9NXxYYqDpDb0ELCn8YfY3K-19Th-2QoA2km/
>
> ――
> View topic http://list.abrf.org/r/topic/12CdQqUzuKr6RluS6gKEIY
> Leave group <email obscured>?Subject=Unsubscribe
>
>

Rest of post

unsubscribe

On Thu, 18 Mar 2021 at 08:06, Mary.Winn <email obscured>> wrote:

> ― 1 file link ―
>
> The Midwest Association of Core Directors is inviting you to share what it
> is like to work in a core and how you got to a career in cores at our April
> outreach event, Why a Career in Cores: Exploring Career Opportunities in
> Shared Research Resource Facilities.
>
> Directed at community college, undergraduate, and graduate students. The
> purpose is to expose scientists to careers in shared research resources and
> the variety of paths available. Will you or your fellow colleagues
> volunteer to share your stories? Register to volunteer at
> https://bit.ly/3ePEv2Y.
>
> We need you to spread the word too! Students and student advocates
> (professors, high school teachers, mentors, coaches, etc.) are able to
> register at the event website: https://mwacd.abrf.org/career-in-cores/.
> An event flyer is attached. Would you send it to at least five colleagues?
>
> This event is not limited to the Midwest. Students and volunteers from
> across the US, Canada, and beyond are encouraged to participate.
>
> Hope to see you there!
> ― 1 file ―
>
>   □ MWACD - Why a Career in Cores.pdf  (276kb)
>
> http://list.abrf.org/r/file/uy1Fl9NXxYYqDpDb0ELCn8YfY3K-19Th-2QoA2km/
>
> ――
> View topic http://list.abrf.org/r/topic/12CdQqUzuKr6RluS6gKEIY
> Leave group <email obscured>?Subject=Unsubscribe
>
>

The Midwest Association of Core Directors is inviting you to share what it is
like to work in a core and how you got to a career in cores at our April
outreach event, Why a Career in Cores: Exploring Career Opportunities in Shared
Research Resource Facilities.

Directed at community college, undergraduate, and graduate students. The
purpose is to expose scientists to careers in shared research resources and the
variety of paths available. Will you or your fellow colleagues volunteer to
share your stories? Register to volunteer at https://bit.ly/3ePEv2Y.

We need you to spread the word too! Students and student advocates (professors,
high school teachers, mentors, coaches, etc.) are able to register at the event
website: https://mwacd.abrf.org/career-in-cores/. An event flyer is attached.
Would you send it to at least five colleagues?

This event is not limited to the Midwest. Students and volunteers from across
the US, Canada, and beyond are encouraged to participate.

Hope to see you there!

MWACD - Why a Career in Cores.pdf 269.9kb

Thank you all for your feedback and suggestions!

Rest of post

Best,

Adam

Hi Adam,
We recommend our users to covalently immobilise the antibody on the beads, and
this allows us to elute with 5% SDS, then use S-trap
(www.protifi.com<http://www.protifi.com>) for digestion/cleanup. Works great.
For biotin-avidin pulldowns, we use on-bead digestion.

Good luck,

Yishai


On 13 Mar 2021, at 3:27, Adam Rabalski
<email obscured><email obscured>>> wrote:

Hi Everyone,

What are you experiences with low pH elution for immuno-precipitation of
proteins from cell lysates? Ideally would like to be able to proceed
immediately after elution/neutralization with in-solution digest and circumvent
any necessary clean up steps (with SP3 or CF/MeOH) that would come with
denaturing conditions like SDS. Low pH elution does not appear to be very
efficient given the amount of protein remaining in the bead fraction.

Best,

Adam
――
View topic http://list.abrf.org/r/topic/5aNqOmXO6cLZxdvtg6eazU
Leave group <email obscured>?Subject=Unsubscribe

Hi all,

Agreeing with Adam and Karl, that eluting IPs with pH or biotin or soluble
peptide, then somehow prepping the eluates for digest, that's a really hard
way to do things.

In addition to Karl's approach for on-bead digestion, we often elute IP
beads with 5 volumes of nonreducing buffered 8M urea, then proceed directly
to alkylation, dilution, & digestion.  For this, we teach clients to use
only 10 ul of IP beads, so that we can elute with 50 ul urea, so that we
can do things in one microtube.   Per Karl's "careful washing" point, it's
essential to drill clients that the last two IP washes cannot contain any
detergent: I like PIPES-buffered saline.  Also, clients need to give the
washed beads one last spin, and then remove every bit of wash buffer with a
gel-loading tip, so that the urea doesn't get diluted.  Urea elution is
good for covalently immobilized IgG, or when folks are using small amounts
of IgG that's not covalently coupled, or for beads that have a lot of
covalent capture protein like SA.  Note that you need to use non-reducing
urea, or the IgG chains will come apart and elute.

I've considered SDS and S-traps, but I worry about recovery of the tiny
amounts of protein expected for an IP.  Alternatively, I suspect that
boiling SDC would be an awesome approach, but I haven't tried it.  ????

But if the antibody is not covalently bound to the beads and there's a lot
of it, urea will elute the IgG, and then the IgG peptides can overwhelm the
nanocolumn.  So when using a lot of non-covalent IgG, we go with gel-LC so
that we can segregate the IgG chains.  Tedious, but works really well when
there's a lot of IgG.  One of our clients preferred this to urea elution,
citing more efficient elution.

Many clients approach their IPs with the expectation that their plans for
specific capture and specific elution will suffice to address specificity.
But the elutions above are not specific.  And in my experience, even the
best IPs are super-noisy.  So we try to get clients to expect that 100's of
proteins will bind non-specifically, and that they must do rigorous
parallel negcon IPs, and think about the experiment as a quantitative
relative-binding assay.

Cheers!

Steve


On Sat, Mar 13, 2021 at 3:55 AM Mechtler,Karl <email obscured>>
wrote:

Rest of post

> Dear Adam,
>
> In my experience, low pH elution does not work in 50% of all cases.
> Therefore, many groups have switched to digesting the peptides directly
> from the beads.
>
> In our group, for example, we perform a 3 h digestion with Lys C on the
> beads. The supernatant is then digested overnight with trypsin. If the
> beads are washed carefully, a desalting step can be saved by working with a
> precolumn.
>
> Enjoy your weekend,
> Karl
>
> -----Ursprüngliche Nachricht-----
> Von: ABRF Discussion Forum <email obscured>> Im Auftrag von Adam
> Rabalski
> Gesendet: Samstag, 13. März 2021 02:28
> An: <email obscured>
> Betreff: [ABRF Discussion Forum] IP Elution Conditions
>
> Hi Everyone,
>
> What are you experiences with low pH elution for immuno-precipitation of
> proteins from cell lysates? Ideally would like to be able to proceed
> immediately after elution/neutralization with in-solution digest and
> circumvent any necessary clean up steps (with SP3 or CF/MeOH) that would
> come with denaturing conditions like SDS. Low pH elution does not appear to
> be very efficient given the amount of protein remaining in the bead
> fraction.
>
> Best,
>
> Adam
> ――
> View topic http://list.abrf.org/r/topic/5aNqOmXO6cLZxdvtg6eazU
> Leave group <email obscured>?Subject=Unsubscribe
>
>
> ――
> View topic http://list.abrf.org/r/topic/7oieQlFNld95iDLs7FtsqE
> Leave group <email obscured>?Subject=Unsubscribe
>
>

--

Steven D. Hartson, Ph.D.
Director, Recombinant DNA/Protein Core Facility
Research Associate Professor
Dept. Biochemistry and Molecular Biology
Oklahoma State University
NRC 246
Stillwater, OK  74078-3035
(405)744-6191 phone
(405)744-7799 FAX
https://genomics.okstate.edu/

Dear Adam,

In my experience, low pH elution does not work in 50% of all cases. Therefore,
many groups have switched to digesting the peptides directly from the beads.

In our group, for example, we perform a 3 h digestion with Lys C on the beads.
The supernatant is then digested overnight with trypsin. If the beads are
washed carefully, a desalting step can be saved by working with a precolumn.

Enjoy your weekend,
Karl

Rest of post

-----Ursprüngliche Nachricht-----
Von: ABRF Discussion Forum <email obscured>> Im Auftrag von Adam Rabalski
Gesendet: Samstag, 13. März 2021 02:28
An: <email obscured>
Betreff: [ABRF Discussion Forum] IP Elution Conditions

Hi Everyone,

What are you experiences with low pH elution for immuno-precipitation of
proteins from cell lysates? Ideally would like to be able to proceed
immediately after elution/neutralization with in-solution digest and circumvent
any necessary clean up steps (with SP3 or CF/MeOH) that would come with
denaturing conditions like SDS. Low pH elution does not appear to be very
efficient given the amount of protein remaining in the bead fraction.

Best,

Adam
――
View topic http://list.abrf.org/r/topic/5aNqOmXO6cLZxdvtg6eazU
Leave group <email obscured>?Subject=Unsubscribe

Hi Everyone,

What are you experiences with low pH elution for immuno-precipitation of
proteins from cell lysates? Ideally would like to be able to proceed
immediately after elution/neutralization with in-solution digest and circumvent
any necessary clean up steps (with SP3 or CF/MeOH) that would come with
denaturing conditions like SDS. Low pH elution does not appear to be very
efficient given the amount of protein remaining in the bead fraction.

Rest of post

Best,

Adam

Wow, the ink is barely dry on that publication.  Thanks Jon.

Best regards,
Brian

Rest of post

________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of J.D.Humphries
<email obscured>>
Sent: Friday, March 12, 2021 4:52 AM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] membrane protein enrichment

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.

Yes. Very interesting thread. Thanks.

I happened to notice this paper in my Google Scholar notifications this morning
and thought it was worth sharing:

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling
https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpubs.acs.org%2Fdoi%2Ffull%2F10.1021%2Facs.analchem.0c04970&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C06345ffb4f1847814d4308d8e53c9727%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637511396004036320%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=riiOlezRGDO0ilGbamjy66N0a90ztx2w8LWqthBu6h0%3D&amp;reserved=0

BW
Jon

Yes. Very interesting thread. Thanks.

I happened to notice this paper in my Google Scholar notifications this morning
and thought it was worth sharing:

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling
https://pubs.acs.org/doi/full/10.1021/acs.analchem.0c04970

Rest of post

BW
Jon

What a great read this morning!
Thank you Brian and Andy for sharing this paper.

Although retired, I very much appreciate (occasionally) reading current
techniques, especially those that build on and refine technique.

Ammonium sulfate cuts of detergent solubilized beef heart cytochrome c oxidase
preparations and lipid head group identification of the remaining tightly bound
lipids was a large part of my first 10 years in science labs (we are talking
the mid '70's to '80's here. The scale of material we worked on with
ultracentrifugation rotors was so different than where we are today. It has
been fantastic to see such progress!

Thanks for the read with my morning coffee!

Deb McMillen



A long-time helpful contributor to this list, Andy Alpert, made me aware of
this paper which is yet another very interesting approach to isolating membrane
proteins. For my purposes (bottom up proteomics), the use of Triton X114 isn't
a problem because removal is easily achieved after digestion using SDB-RPS or
similar tips in HILIC/SCX mode as described in the EasyPhos Nature Methods
paper.

Thanks Andy for your input!

"Top-Down Proteomics of Endogenous Membrane Proteins Enabled by Cloud Point
Enrichment and Multidimensional Liquid Chromatography−Mass Spectrometry"
[ https://dx.doi.org/10.1021/acs.analchem.0c02533 |
https://dx.doi.org/10.1021/acs.analchem.0c02533 ]

Best regards,
Brian

A long-time helpful contributor to this list,  Andy Alpert, made me aware of
this paper which is yet another very interesting approach to isolating membrane
proteins.  For my purposes (bottom up proteomics), the use of Triton X114 isn't
a problem because removal is easily achieved after digestion using SDB-RPS or
similar tips in HILIC/SCX mode as described in the EasyPhos Nature Methods
paper.

Thanks Andy for your input!

"Top-Down Proteomics of Endogenous Membrane Proteins Enabled by Cloud Point
Enrichment and Multidimensional Liquid Chromatography−Mass Spectrometry"
https://dx.doi.org/10.1021/acs.analchem.0c02533

Best regards,
Brian

Rest of post


________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Yan Wang
<email obscured>>
Sent: Wednesday, March 10, 2021 10:02 AM
To: ABRF Discussion List <email obscured>>
Subject: Re: [ABRF Discussion Forum] membrane protein enrichment

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.



Thanks to everyone for the wonderful responses.

Brian, your hijacking is much appreciated. I'm learning more than I
bargained for.

Kim, if I'm not mistaken, what you are proposing is to biotinylate cell
surface proteins followed by lysing, and digestion, then immuno enrichment?
How does biotinylation affect trypsin activity? Then I suppose we get into
how many peptides do we get from each protein for ID?

Overall, it looks like there's no consensus on product or protocol. I'll
have to talk with my collaborator to see how to proceed.

Yan

On Wed, Mar 10, 2021 at 9:31 AM Hampton, Brian <email obscured>>
wrote:

> Hello Bill,
>
> I'm curious as to the extent of biotinylation observed in the data.  Was
> the Lys modification CAMThiopropyl (after release from the resin by
> reduction) commonly found on the membrane proteins identified in your
> samples?
>
> Best regards,
> Brian
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of William
> Breuer <email obscured>>
> Sent: Tuesday, March 9, 2021 9:36 AM
> To: <email obscured> <email obscured>>
> Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
>
> 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.
>
>
>
> Hi,
> we had reasonable success with the Sulfo-NHS-SS-Biotin cell surface
> labeling kit from Thermo (but I think the kit is actually from Pierce).
> It's called EZ-link-Sulfo-NHS-SS-Biotin, Product # 21331. We ID'd a total
> of 485 proteins, out of which 197 were cell-surface or extracellular
> proteins.
>
> Bill Breuer
> Proteomics/Mass spectrometry Unit,
> Room 1-453, 4th floor, wing 1,
> Institute of Life Sciences,
> Edmond J. Safra Campus - Givat Ram,
> Hebrew University of Jerusalem,
> Jerusalem, 9190401
> Israel
>
> On Tue, Mar 9, 2021 at 1:57 PM Hampton, Brian <email obscured>>
> wrote:
>
> > Hi Yan,
> >
> > I am sorry for not correctly reading your email.  My mistake.
> >
> > I am in the process of trying to analyze the cell membrane proteome of
> two
> > related cell types.  I have come across two different approaches.  One is
> > protease shaving and the other is classic surface biotinylation.
> >
> > The main problem I have with cell surface biotinylation is the large
> > background of cytosolic and nuclear proteins that are nonspecifically
> > captured on the avidin beads.  I'm using Sulfo-NHS-SS-Biotin to label the
> > cells followed by reduction with TCEP to release from the avidin beads.
> > I'm trying different pull-down matrices in hopes of finding one with less
> > non-specific binding.  I'm just using the literature to cobble together a
> > protocol.  One step I want to try next, I have not seen in the
> literature,
> > is to incorporate a ~harshish wash of the beads before release of the
> > biotin bound material.  This could help reduce the non-specific that is
> > recovered following reduction.  I was using a mild detergent initially
> > hoping to also pick up interacting proteins.
> >
> > I did begin the other method of using a protease to shave off proteins
> > exposed on the cell surface.  But it got messy.  I ended up with a fairly
> > large volume and I worried that the method in general would not be
> > reproducible enough to get accurate results for comparisons.
> >
> > If you are looking for a kit, I think Pierce may have this for surface
> > biotin labeling.  I came across a poster of theirs on this topic, but I
> > didn't follow up to check availability.
> >
> > Best regards,
> > Brian
> > ________________________________
> > From: ABRF Discussion Forum <email obscured>> on behalf of Yan Wang <
> > <email obscured>>
> > Sent: Sunday, March 7, 2021 11:21 PM
> > To: ABRF Discussion List <email obscured>>
> > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> >
> > 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.
> >
> >
> >
> > Sorry I was not clear. I'm looking for protocols or products for
> enrichment
> > or isolation or cell membrane proteins. Any suggestions?
> > Yan
> >
> > On Sun, Mar 7, 2021 at 1:14 PM Hampton, Brian <
> <email obscured>>
> > wrote:
> >
> > > Hello,
> > >
> > > Do you mean molecular weight cutoff membrane filter concentrators or
> some
> > > form of affinity or charge derivatized membrane for enrichment?  I have
> > > experience with the former but not the latter.  If the latter, I would
> > like
> > > to know what you are considering.
> > >
> > > Thanks,
> > > Brian
> > >
> > > ________________________________
> > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> Wang <
> > > <email obscured>>
> > > Sent: Friday, March 5, 2021 6:23 PM
> > > To: ABRF Discussion List <email obscured>>
> > > Subject: [ABRF Discussion Forum] membrane protein enrichment
> > >
> > > 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.
> > >
> > >
> > >
> > > >
> > > > I'm wondering if folks here have good or bad experience with membrane
> > > > protein enrichment products?
> > > >
> > >
> > > Yan
> > >
> > > ――
> > > View topic
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2Fj2lKnx6hjbX8afQsUHxrU&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342259358%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=vOyAFvV5xje9yIF%2F58CeYTjVS98tFT%2FiHCPSXD0MsNg%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> > > ――
> > > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F1z9BOZpuxXALLkxUV4GS0P&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342259358%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=AsmPspq7LI2W%2BRiLu%2FtZvSRgBGfY3XL4UYnvGnlojRw%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> >
> > ――
> > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F33lvNigwYjq9uGyBYvQwOY&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342259358%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=ozA4U8Oar%2BvbN5aJR%2FgUujK85vrY0zk8Oneyx0ileiQ%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
> > ――
> > View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4bFEJxyuMn3rbCgZCxkE8n&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342269348%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=BzeoZxQ1sYpe7Jpn%2F2F%2BPh1ZzeCXNeXGChXO7KPVH7w%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
>
> ――
> View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F21tFDW3Sw8V7G0ypVTSCBr&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342269348%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=rWD%2FTe%2FoMdlqIvGL0KayixA1eM%2Fa8IpEdQfO2XiopNI%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>
> ――
> View topic
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4b9AtSgevrPLwArjSv15CM&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342269348%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=BWMYmyAysQZhrYcKswsnmZdI9X01yKSsyDS02gMP35E%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>

――
View topic
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F41MHi93eh7btJ8EtgVkdo&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2d3c38e62e754cc5ebe808d8e3d5a373%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510636342269348%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=FhzPgBvVzOpFVfRUPmFAkS1%2BRkI2l1PLpuKiLJSh1W4%3D&amp;reserved=0
Leave group <email obscured>?Subject=Unsubscribe

Thanks Bill,

I searched with and without the CAMthiopropyl (alkylated form of the cleaved
moiety) and was surprised that only 20 or so peptides were picked up with the
modification.  I was expecting a lot of lysine modification but that didn't
seem to be the case.

Thanks again for your input.

Rest of post

Brian
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of William Breuer
<email obscured>>
Sent: Wednesday, March 10, 2021 10:05 AM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] membrane protein enrichment

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.



Hi Brian,
I found only about 20 such proteins per sample with Maxquant, but the
search was complicated by the fact that after the release of the proteins
using DTT we alkylated with iodoacetamide, which presumably also reacted
with the biotin-SH moiety. I was not absolutely sure that I specified the
correct MW of the final derivative, so it was not conclusive. But the
enrichment of cell surface and external proteins was significant.
Bill

On Wed, Mar 10, 2021 at 4:31 PM Hampton, Brian <email obscured>>
wrote:

> Hello Bill,
>
> I'm curious as to the extent of biotinylation observed in the data.  Was
> the Lys modification CAMThiopropyl (after release from the resin by
> reduction) commonly found on the membrane proteins identified in your
> samples?
>
> Best regards,
> Brian
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of William
> Breuer <email obscured>>
> Sent: Tuesday, March 9, 2021 9:36 AM
> To: <email obscured> <email obscured>>
> Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
>
> 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.
>
>
>
> Hi,
> we had reasonable success with the Sulfo-NHS-SS-Biotin cell surface
> labeling kit from Thermo (but I think the kit is actually from Pierce).
> It's called EZ-link-Sulfo-NHS-SS-Biotin, Product # 21331. We ID'd a total
> of 485 proteins, out of which 197 were cell-surface or extracellular
> proteins.
>
> Bill Breuer
> Proteomics/Mass spectrometry Unit,
> Room 1-453, 4th floor, wing 1,
> Institute of Life Sciences,
> Edmond J. Safra Campus - Givat Ram,
> Hebrew University of Jerusalem,
> Jerusalem, 9190401
> Israel
>
> On Tue, Mar 9, 2021 at 1:57 PM Hampton, Brian <email obscured>>
> wrote:
>
> > Hi Yan,
> >
> > I am sorry for not correctly reading your email.  My mistake.
> >
> > I am in the process of trying to analyze the cell membrane proteome of
> two
> > related cell types.  I have come across two different approaches.  One is
> > protease shaving and the other is classic surface biotinylation.
> >
> > The main problem I have with cell surface biotinylation is the large
> > background of cytosolic and nuclear proteins that are nonspecifically
> > captured on the avidin beads.  I'm using Sulfo-NHS-SS-Biotin to label the
> > cells followed by reduction with TCEP to release from the avidin beads.
> > I'm trying different pull-down matrices in hopes of finding one with less
> > non-specific binding.  I'm just using the literature to cobble together a
> > protocol.  One step I want to try next, I have not seen in the
> literature,
> > is to incorporate a ~harshish wash of the beads before release of the
> > biotin bound material.  This could help reduce the non-specific that is
> > recovered following reduction.  I was using a mild detergent initially
> > hoping to also pick up interacting proteins.
> >
> > I did begin the other method of using a protease to shave off proteins
> > exposed on the cell surface.  But it got messy.  I ended up with a fairly
> > large volume and I worried that the method in general would not be
> > reproducible enough to get accurate results for comparisons.
> >
> > If you are looking for a kit, I think Pierce may have this for surface
> > biotin labeling.  I came across a poster of theirs on this topic, but I
> > didn't follow up to check availability.
> >
> > Best regards,
> > Brian
> > ________________________________
> > From: ABRF Discussion Forum <email obscured>> on behalf of Yan Wang <
> > <email obscured>>
> > Sent: Sunday, March 7, 2021 11:21 PM
> > To: ABRF Discussion List <email obscured>>
> > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> >
> > 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.
> >
> >
> >
> > Sorry I was not clear. I'm looking for protocols or products for
> enrichment
> > or isolation or cell membrane proteins. Any suggestions?
> > Yan
> >
> > On Sun, Mar 7, 2021 at 1:14 PM Hampton, Brian <
> <email obscured>>
> > wrote:
> >
> > > Hello,
> > >
> > > Do you mean molecular weight cutoff membrane filter concentrators or
> some
> > > form of affinity or charge derivatized membrane for enrichment?  I have
> > > experience with the former but not the latter.  If the latter, I would
> > like
> > > to know what you are considering.
> > >
> > > Thanks,
> > > Brian
> > >
> > > ________________________________
> > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> Wang <
> > > <email obscured>>
> > > Sent: Friday, March 5, 2021 6:23 PM
> > > To: ABRF Discussion List <email obscured>>
> > > Subject: [ABRF Discussion Forum] membrane protein enrichment
> > >
> > > 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.
> > >
> > >
> > >
> > > >
> > > > I'm wondering if folks here have good or bad experience with membrane
> > > > protein enrichment products?
> > > >
> > >
> > > Yan
> > >
> > > ――
> > > View topic
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2Fj2lKnx6hjbX8afQsUHxrU&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154108122%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=iAMXz0L7dE7qc4C65lWdKn7p1YaCq4M7g8Bvw2AjK8I%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> > > ――
> > > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F1z9BOZpuxXALLkxUV4GS0P&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154118120%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=dv5tpbt66jkT3bO0cl%2FhXFIo5hsHU%2FT8JmqEFYTBKYQ%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> >
> > ――
> > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F33lvNigwYjq9uGyBYvQwOY&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154118120%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=TY2pkBnN25T86Nqh8SjDtWWgbyJRNrUvE3udb6o8kLs%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
> > ――
> > View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4bFEJxyuMn3rbCgZCxkE8n&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154118120%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=tXl%2FdolLjhKlp8UQDSSMQVtH7%2FcEknDpwT6HfCyVAmc%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
>
> ――
> View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F21tFDW3Sw8V7G0ypVTSCBr&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154118120%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=mn1R8Rh9JmO6ntlU0JX%2B8EP3%2Fam4ls6UR6FlyxpKLtU%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>
> ――
> View topic
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4b9AtSgevrPLwArjSv15CM&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154118120%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=BP6d3%2B1XWBsulwZF24dCSDju7BUOKTQBNwxzW8ICBvY%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>

――
View topic
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F7Ediun13fKJkNmugADr72H&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7Caeeba335534c46193c1908d8e3d60f73%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637510637154118120%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=1J62zSbOr8UfPhe4ltwH2u3SN7B9IvxxvBc1Zb5H%2Bpo%3D&amp;reserved=0
Leave group <email obscured>?Subject=Unsubscribe

ABRF is pleased to share information on this upcoming webinar presented by CTLS

This webinar will be hosted in a collaboration between Core Technologies for
Life Sciences (CTLS) and the Association of Biomolecular Resource Facilities
(ABRF) co-moderated by Josh Rappoport (Boston College) and Andy Chitty (OHSU).
Speakers from different continents will present exciting views into distinct
core facility operating models from across the globe.  Ranging in size, scope,
userbase, and funding sources, the world of core facilities spans a wide range
of operating models, and this session will provide insights into diverse
organizational structures, as well as the common challenges that all cores must
face.

https://ctls-org.eu/events/march-session-core-facility-operating-models-across-the-globe/

Hi Yan,

I agree, this has been a very informative discussion topic!
Yes, I'm suggesting exactly what you said, biotinylating surface proteins,
washing, lysis, digestion, peptide cleanup, and immunoenrichment at the
peptide level.  Trypsin shouldn't cleave at biotinylated residues, so
peptides may be longer than average.  The number of peptides per protein
will depend on amount of the protein that is cell surface-exposed and the
efficiency of biotinylation of that protein.  As with any
modification-based enrichment, assessing FDR at the peptide rather than
protein level may be more appropriate.

Rest of post

Best,
Kim

On Wed, Mar 10, 2021 at 10:03 AM Yan Wang <email obscured>> wrote:

> Thanks to everyone for the wonderful responses.
>
> Brian, your hijacking is much appreciated. I'm learning more than I
> bargained for.
>
> Kim, if I'm not mistaken, what you are proposing is to biotinylate cell
> surface proteins followed by lysing, and digestion, then immuno enrichment?
> How does biotinylation affect trypsin activity? Then I suppose we get into
> how many peptides do we get from each protein for ID?
>
> Overall, it looks like there's no consensus on product or protocol. I'll
> have to talk with my collaborator to see how to proceed.
>
> Yan
>
> On Wed, Mar 10, 2021 at 9:31 AM Hampton, Brian <email obscured>
> >
> wrote:
>
> > Hello Bill,
> >
> > I'm curious as to the extent of biotinylation observed in the data.  Was
> > the Lys modification CAMThiopropyl (after release from the resin by
> > reduction) commonly found on the membrane proteins identified in your
> > samples?
> >
> > Best regards,
> > Brian
> > ________________________________
> > From: ABRF Discussion Forum <email obscured>> on behalf of William
> > Breuer <email obscured>>
> > Sent: Tuesday, March 9, 2021 9:36 AM
> > To: <email obscured> <email obscured>>
> > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> >
> > 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.
> >
> >
> >
> > Hi,
> > we had reasonable success with the Sulfo-NHS-SS-Biotin cell surface
> > labeling kit from Thermo (but I think the kit is actually from Pierce).
> > It's called EZ-link-Sulfo-NHS-SS-Biotin, Product # 21331. We ID'd a total
> > of 485 proteins, out of which 197 were cell-surface or extracellular
> > proteins.
> >
> > Bill Breuer
> > Proteomics/Mass spectrometry Unit,
> > Room 1-453, 4th floor, wing 1,
> > Institute of Life Sciences,
> > Edmond J. Safra Campus - Givat Ram,
> > Hebrew University of Jerusalem,
> > Jerusalem, 9190401
> > Israel
> >
> > On Tue, Mar 9, 2021 at 1:57 PM Hampton, Brian <
> <email obscured>>
> > wrote:
> >
> > > Hi Yan,
> > >
> > > I am sorry for not correctly reading your email.  My mistake.
> > >
> > > I am in the process of trying to analyze the cell membrane proteome of
> > two
> > > related cell types.  I have come across two different approaches.  One
> is
> > > protease shaving and the other is classic surface biotinylation.
> > >
> > > The main problem I have with cell surface biotinylation is the large
> > > background of cytosolic and nuclear proteins that are nonspecifically
> > > captured on the avidin beads.  I'm using Sulfo-NHS-SS-Biotin to label
> the
> > > cells followed by reduction with TCEP to release from the avidin beads.
> > > I'm trying different pull-down matrices in hopes of finding one with
> less
> > > non-specific binding.  I'm just using the literature to cobble
> together a
> > > protocol.  One step I want to try next, I have not seen in the
> > literature,
> > > is to incorporate a ~harshish wash of the beads before release of the
> > > biotin bound material.  This could help reduce the non-specific that is
> > > recovered following reduction.  I was using a mild detergent initially
> > > hoping to also pick up interacting proteins.
> > >
> > > I did begin the other method of using a protease to shave off proteins
> > > exposed on the cell surface.  But it got messy.  I ended up with a
> fairly
> > > large volume and I worried that the method in general would not be
> > > reproducible enough to get accurate results for comparisons.
> > >
> > > If you are looking for a kit, I think Pierce may have this for surface
> > > biotin labeling.  I came across a poster of theirs on this topic, but I
> > > didn't follow up to check availability.
> > >
> > > Best regards,
> > > Brian
> > > ________________________________
> > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> Wang <
> > > <email obscured>>
> > > Sent: Sunday, March 7, 2021 11:21 PM
> > > To: ABRF Discussion List <email obscured>>
> > > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> > >
> > > 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.
> > >
> > >
> > >
> > > Sorry I was not clear. I'm looking for protocols or products for
> > enrichment
> > > or isolation or cell membrane proteins. Any suggestions?
> > > Yan
> > >
> > > On Sun, Mar 7, 2021 at 1:14 PM Hampton, Brian <
> > <email obscured>>
> > > wrote:
> > >
> > > > Hello,
> > > >
> > > > Do you mean molecular weight cutoff membrane filter concentrators or
> > some
> > > > form of affinity or charge derivatized membrane for enrichment?  I
> have
> > > > experience with the former but not the latter.  If the latter, I
> would
> > > like
> > > > to know what you are considering.
> > > >
> > > > Thanks,
> > > > Brian
> > > >
> > > > ________________________________
> > > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> > Wang <
> > > > <email obscured>>
> > > > Sent: Friday, March 5, 2021 6:23 PM
> > > > To: ABRF Discussion List <email obscured>>
> > > > Subject: [ABRF Discussion Forum] membrane protein enrichment
> > > >
> > > > 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.
> > > >
> > > >
> > > >
> > > > >
> > > > > I'm wondering if folks here have good or bad experience with
> membrane
> > > > > protein enrichment products?
> > > > >
> > > >
> > > > Yan
> > > >
> > > > ――
> > > > View topic
> > > >
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2Fj2lKnx6hjbX8afQsUHxrU&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999128441%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=V3IrTGCfF385EizajM7D5%2FueuK7xItqqD81h5T1qL5s%3D&amp;reserved=0
> > > > Leave group <email obscured>?Subject=Unsubscribe
> > > >
> > > >
> > > > ――
> > > > View topic
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F1z9BOZpuxXALLkxUV4GS0P&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999128441%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=0A%2F150WO2nqryfEm%2F8LkcKlMRNLS8npYi9eUZOv12vc%3D&amp;reserved=0
> > > > Leave group <email obscured>?Subject=Unsubscribe
> > > >
> > > >
> > >
> > > ――
> > > View topic
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F33lvNigwYjq9uGyBYvQwOY&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=rFHV9IGhKiR1n3HQ0PzPWQB7Ge2At3XaORk7DdEIc1E%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> > > ――
> > > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4bFEJxyuMn3rbCgZCxkE8n&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=jmgblqR9rKltSuPPu%2FDPDm%2F%2BfGcX9MBZebCcoEHrRWc%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> >
> > ――
> > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F21tFDW3Sw8V7G0ypVTSCBr&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=vnZvYLxoRwgIqGQmvCYtycjJK0NVEPA5y7Fr3t8tW8E%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
> > ――
> > View topic http://list.abrf.org/r/topic/4b9AtSgevrPLwArjSv15CM
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
>
> ――
> View topic http://list.abrf.org/r/topic/41MHi93eh7btJ8EtgVkdo
> Leave group <email obscured>?Subject=Unsubscribe
>
>

--
This message contains information which may be confidential and/or
protected by attorney-client privilege.  Unless you are the addressee, you
may not use, copy or disclose to anyone this message or any information
contained in this message.  If you have received this message in error,
please send me an email and delete this message.   Thank you.

Hi Yan,

Our lab published a paper a few years ago. In our hands/experiences,  the
enrichment approach via membrane isolation/centrifugation was better than the
cell surface biotinylation approach. Please look at the reference paper for
detail
 https://pubmed.ncbi.nlm.nih.gov/28116781/

Rest of post

Best,

Hua

-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Yan Wang
Sent: Wednesday, March 10, 2021 10:02 AM
To: ABRF Discussion List <email obscured>>
Subject: [External] Re: [ABRF Discussion Forum] membrane protein enrichment

Thanks to everyone for the wonderful responses.

Brian, your hijacking is much appreciated. I'm learning more than I bargained
for.

Kim, if I'm not mistaken, what you are proposing is to biotinylate cell surface
proteins followed by lysing, and digestion, then immuno enrichment?
How does biotinylation affect trypsin activity? Then I suppose we get into how
many peptides do we get from each protein for ID?

Overall, it looks like there's no consensus on product or protocol. I'll have
to talk with my collaborator to see how to proceed.

Yan

On Wed, Mar 10, 2021 at 9:31 AM Hampton, Brian <email obscured>>
wrote:

> Hello Bill,
>
> I'm curious as to the extent of biotinylation observed in the data.
> Was the Lys modification CAMThiopropyl (after release from the resin
> by
> reduction) commonly found on the membrane proteins identified in your
> samples?
>
> Best regards,
> Brian
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of William
> Breuer <email obscured>>
> Sent: Tuesday, March 9, 2021 9:36 AM
> To: <email obscured> <email obscured>>
> Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
>
> 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.
>
>
>
> Hi,
> we had reasonable success with the Sulfo-NHS-SS-Biotin cell surface
> labeling kit from Thermo (but I think the kit is actually from Pierce).
> It's called EZ-link-Sulfo-NHS-SS-Biotin, Product # 21331. We ID'd a
> total of 485 proteins, out of which 197 were cell-surface or
> extracellular proteins.
>
> Bill Breuer
> Proteomics/Mass spectrometry Unit,
> Room 1-453, 4th floor, wing 1,
> Institute of Life Sciences,
> Edmond J. Safra Campus - Givat Ram,
> Hebrew University of Jerusalem,
> Jerusalem, 9190401
> Israel
>
> On Tue, Mar 9, 2021 at 1:57 PM Hampton, Brian
> <email obscured>>
> wrote:
>
> > Hi Yan,
> >
> > I am sorry for not correctly reading your email.  My mistake.
> >
> > I am in the process of trying to analyze the cell membrane proteome
> > of
> two
> > related cell types.  I have come across two different approaches.
> > One is protease shaving and the other is classic surface biotinylation.
> >
> > The main problem I have with cell surface biotinylation is the large
> > background of cytosolic and nuclear proteins that are
> > nonspecifically captured on the avidin beads.  I'm using
> > Sulfo-NHS-SS-Biotin to label the cells followed by reduction with TCEP to
release from the avidin beads.
> > I'm trying different pull-down matrices in hopes of finding one with
> > less non-specific binding.  I'm just using the literature to cobble
> > together a protocol.  One step I want to try next, I have not seen
> > in the
> literature,
> > is to incorporate a ~harshish wash of the beads before release of
> > the biotin bound material.  This could help reduce the non-specific
> > that is recovered following reduction.  I was using a mild detergent
> > initially hoping to also pick up interacting proteins.
> >
> > I did begin the other method of using a protease to shave off
> > proteins exposed on the cell surface.  But it got messy.  I ended up
> > with a fairly large volume and I worried that the method in general
> > would not be reproducible enough to get accurate results for comparisons.
> >
> > If you are looking for a kit, I think Pierce may have this for
> > surface biotin labeling.  I came across a poster of theirs on this
> > topic, but I didn't follow up to check availability.
> >
> > Best regards,
> > Brian
> > ________________________________
> > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> > Wang < <email obscured>>
> > Sent: Sunday, March 7, 2021 11:21 PM
> > To: ABRF Discussion List <email obscured>>
> > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> >
> > 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.
> >
> >
> >
> > Sorry I was not clear. I'm looking for protocols or products for
> enrichment
> > or isolation or cell membrane proteins. Any suggestions?
> > Yan
> >
> > On Sun, Mar 7, 2021 at 1:14 PM Hampton, Brian <
> <email obscured>>
> > wrote:
> >
> > > Hello,
> > >
> > > Do you mean molecular weight cutoff membrane filter concentrators
> > > or
> some
> > > form of affinity or charge derivatized membrane for enrichment?  I
> > > have experience with the former but not the latter.  If the
> > > latter, I would
> > like
> > > to know what you are considering.
> > >
> > > Thanks,
> > > Brian
> > >
> > > ________________________________
> > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> Wang <
> > > <email obscured>>
> > > Sent: Friday, March 5, 2021 6:23 PM
> > > To: ABRF Discussion List <email obscured>>
> > > Subject: [ABRF Discussion Forum] membrane protein enrichment
> > >
> > > 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.
> > >
> > >
> > >
> > > >
> > > > I'm wondering if folks here have good or bad experience with
> > > > membrane protein enrichment products?
> > > >
> > >
> > > Yan
> > >
> > > ――
> > > View topic
> > >
> >
> https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.
> abrf.org%2Fr%2Ftopic%2Fj2lKnx6hjbX8afQsUHxrU&amp;data=04%7C01%7CDINGH%
> 40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d1
> 46b575c975%7C1%7C0%7C637509854987507596%7CUnknown%7CTWFpbGZsb3d8eyJWIj
> oiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&am
> p;sdata=p%2FWgRV1PkVC3jezNbE%2BVi%2BkxRZK8fOUj%2FQzxKEa5XOg%3D&amp;res
> erved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> > > ――
> > > View topic
> >
> https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.
> abrf.org%2Fr%2Ftopic%2F1z9BOZpuxXALLkxUV4GS0P&amp;data=04%7C01%7CDINGH
> %40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d
> 146b575c975%7C1%7C0%7C637509854987507596%7CUnknown%7CTWFpbGZsb3d8eyJWI
> joiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&a
> mp;sdata=tDGx0lgA%2Blo3PktjdkZ%2FZaOs2QEUreGuRqRTckxXhZk%3D&amp;reserv
> ed=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> >
> > ――
> > View topic
> >
> https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.
> abrf.org%2Fr%2Ftopic%2F33lvNigwYjq9uGyBYvQwOY&amp;data=04%7C01%7CDINGH
> %40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d
> 146b575c975%7C1%7C0%7C637509854987517548%7CUnknown%7CTWFpbGZsb3d8eyJWI
> joiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&a
> mp;sdata=weMvhbDVBtyTf0AO372SFScF8iRKWsl4vXAo681SDC0%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
> > ――
> > View topic
> https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.
> abrf.org%2Fr%2Ftopic%2F4bFEJxyuMn3rbCgZCxkE8n&amp;data=04%7C01%7CDINGH
> %40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d
> 146b575c975%7C1%7C0%7C637509854987517548%7CUnknown%7CTWFpbGZsb3d8eyJWI
> joiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&a
> mp;sdata=VEGYdWv%2BGcHBOxoVd9dwZao6gmnbos8grnk%2F7qjuUA0%3D&amp;reserv
> ed=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
>
> ――
> View topic
> https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.
> abrf.org%2Fr%2Ftopic%2F21tFDW3Sw8V7G0ypVTSCBr&amp;data=04%7C01%7CDINGH
> %40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d
> 146b575c975%7C1%7C0%7C637509854987517548%7CUnknown%7CTWFpbGZsb3d8eyJWI
> joiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&a
> mp;sdata=GYASmOANRMVaF7MnVU7uIf062ZjwsvU2t%2Fpev1m%2B4y8%3D&amp;reserv
> ed=0 Leave group <email obscured>?Subject=Unsubscribe
>
>
> ――
> View topic
> https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.
> abrf.org%2Fr%2Ftopic%2F4b9AtSgevrPLwArjSv15CM&amp;data=04%7C01%7CDINGH
> %40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d
> 146b575c975%7C1%7C0%7C637509854987517548%7CUnknown%7CTWFpbGZsb3d8eyJWI
> joiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&a
> mp;sdata=X2IK1MJFcthFJldSqsAFDdxDIJAtt8KHw6eEXBgFGK8%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>

――
View topic
https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F41MHi93eh7btJ8EtgVkdo&amp;data=04%7C01%7CDINGH%40chop.edu%7C37e3e38ee4b8453d81bd08d8e3d5dde3%7Ca611241607b041a59bb1d146b575c975%7C1%7C0%7C637509854987517548%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&amp;sdata=QZllj6sD8BARLVSNYrLfxQk0zlypmyXGmUF7SoSalkM%3D&amp;reserved=0
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**.

Hi all,

You might find some tricks useful that are described in D. R. Francoleon, et
al. DOI: 10.1021/pr800923e. We biotinylated surface proteins in archaeal cells,
grown under reducing conditions where the S-S biotinylating reagent couldn’t be
used. Because the cells had to be adapted to cultivate at a pH higher than
normal for labeling, we were especially worried about cell lysis during the
labeling. To overcome that problem, we used silicone oil to separated intact
cells from the labeling mixture, so that intact cells could be harvested
without contamination from lysed cell contents. With the right density oil
(remember density is temperature-dependent) you get three phases.

The study was most interested in identifying the organism’s surface-layer
protein, so stringent washes with 2% SDS were incorporated. While that’s
probably washing more stringently than most studies would want, you get an idea
of what’s possible with the affinity of biotin/streptavidin.

Rest of post

Rachel

Hi Brian,
I found only about 20 such proteins per sample with Maxquant, but the
search was complicated by the fact that after the release of the proteins
using DTT we alkylated with iodoacetamide, which presumably also reacted
with the biotin-SH moiety. I was not absolutely sure that I specified the
correct MW of the final derivative, so it was not conclusive. But the
enrichment of cell surface and external proteins was significant.
Bill

On Wed, Mar 10, 2021 at 4:31 PM Hampton, Brian <email obscured>>
wrote:

Rest of post

> Hello Bill,
>
> I'm curious as to the extent of biotinylation observed in the data.  Was
> the Lys modification CAMThiopropyl (after release from the resin by
> reduction) commonly found on the membrane proteins identified in your
> samples?
>
> Best regards,
> Brian
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of William
> Breuer <email obscured>>
> Sent: Tuesday, March 9, 2021 9:36 AM
> To: <email obscured> <email obscured>>
> Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
>
> 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.
>
>
>
> Hi,
> we had reasonable success with the Sulfo-NHS-SS-Biotin cell surface
> labeling kit from Thermo (but I think the kit is actually from Pierce).
> It's called EZ-link-Sulfo-NHS-SS-Biotin, Product # 21331. We ID'd a total
> of 485 proteins, out of which 197 were cell-surface or extracellular
> proteins.
>
> Bill Breuer
> Proteomics/Mass spectrometry Unit,
> Room 1-453, 4th floor, wing 1,
> Institute of Life Sciences,
> Edmond J. Safra Campus - Givat Ram,
> Hebrew University of Jerusalem,
> Jerusalem, 9190401
> Israel
>
> On Tue, Mar 9, 2021 at 1:57 PM Hampton, Brian <email obscured>>
> wrote:
>
> > Hi Yan,
> >
> > I am sorry for not correctly reading your email.  My mistake.
> >
> > I am in the process of trying to analyze the cell membrane proteome of
> two
> > related cell types.  I have come across two different approaches.  One is
> > protease shaving and the other is classic surface biotinylation.
> >
> > The main problem I have with cell surface biotinylation is the large
> > background of cytosolic and nuclear proteins that are nonspecifically
> > captured on the avidin beads.  I'm using Sulfo-NHS-SS-Biotin to label the
> > cells followed by reduction with TCEP to release from the avidin beads.
> > I'm trying different pull-down matrices in hopes of finding one with less
> > non-specific binding.  I'm just using the literature to cobble together a
> > protocol.  One step I want to try next, I have not seen in the
> literature,
> > is to incorporate a ~harshish wash of the beads before release of the
> > biotin bound material.  This could help reduce the non-specific that is
> > recovered following reduction.  I was using a mild detergent initially
> > hoping to also pick up interacting proteins.
> >
> > I did begin the other method of using a protease to shave off proteins
> > exposed on the cell surface.  But it got messy.  I ended up with a fairly
> > large volume and I worried that the method in general would not be
> > reproducible enough to get accurate results for comparisons.
> >
> > If you are looking for a kit, I think Pierce may have this for surface
> > biotin labeling.  I came across a poster of theirs on this topic, but I
> > didn't follow up to check availability.
> >
> > Best regards,
> > Brian
> > ________________________________
> > From: ABRF Discussion Forum <email obscured>> on behalf of Yan Wang <
> > <email obscured>>
> > Sent: Sunday, March 7, 2021 11:21 PM
> > To: ABRF Discussion List <email obscured>>
> > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> >
> > 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.
> >
> >
> >
> > Sorry I was not clear. I'm looking for protocols or products for
> enrichment
> > or isolation or cell membrane proteins. Any suggestions?
> > Yan
> >
> > On Sun, Mar 7, 2021 at 1:14 PM Hampton, Brian <
> <email obscured>>
> > wrote:
> >
> > > Hello,
> > >
> > > Do you mean molecular weight cutoff membrane filter concentrators or
> some
> > > form of affinity or charge derivatized membrane for enrichment?  I have
> > > experience with the former but not the latter.  If the latter, I would
> > like
> > > to know what you are considering.
> > >
> > > Thanks,
> > > Brian
> > >
> > > ________________________________
> > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> Wang <
> > > <email obscured>>
> > > Sent: Friday, March 5, 2021 6:23 PM
> > > To: ABRF Discussion List <email obscured>>
> > > Subject: [ABRF Discussion Forum] membrane protein enrichment
> > >
> > > 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.
> > >
> > >
> > >
> > > >
> > > > I'm wondering if folks here have good or bad experience with membrane
> > > > protein enrichment products?
> > > >
> > >
> > > Yan
> > >
> > > ――
> > > View topic
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2Fj2lKnx6hjbX8afQsUHxrU&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999128441%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=V3IrTGCfF385EizajM7D5%2FueuK7xItqqD81h5T1qL5s%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> > > ――
> > > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F1z9BOZpuxXALLkxUV4GS0P&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999128441%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=0A%2F150WO2nqryfEm%2F8LkcKlMRNLS8npYi9eUZOv12vc%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> >
> > ――
> > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F33lvNigwYjq9uGyBYvQwOY&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=rFHV9IGhKiR1n3HQ0PzPWQB7Ge2At3XaORk7DdEIc1E%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
> > ――
> > View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4bFEJxyuMn3rbCgZCxkE8n&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=jmgblqR9rKltSuPPu%2FDPDm%2F%2BfGcX9MBZebCcoEHrRWc%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
>
> ――
> View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F21tFDW3Sw8V7G0ypVTSCBr&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=vnZvYLxoRwgIqGQmvCYtycjJK0NVEPA5y7Fr3t8tW8E%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>
> ――
> View topic http://list.abrf.org/r/topic/4b9AtSgevrPLwArjSv15CM
> Leave group <email obscured>?Subject=Unsubscribe
>
>

Thanks to everyone for the wonderful responses.

Brian, your hijacking is much appreciated. I'm learning more than I
bargained for.

Kim, if I'm not mistaken, what you are proposing is to biotinylate cell
surface proteins followed by lysing, and digestion, then immuno enrichment?
How does biotinylation affect trypsin activity? Then I suppose we get into
how many peptides do we get from each protein for ID?

Overall, it looks like there's no consensus on product or protocol. I'll
have to talk with my collaborator to see how to proceed.

Yan

On Wed, Mar 10, 2021 at 9:31 AM Hampton, Brian <email obscured>>
wrote:

Rest of post

> Hello Bill,
>
> I'm curious as to the extent of biotinylation observed in the data.  Was
> the Lys modification CAMThiopropyl (after release from the resin by
> reduction) commonly found on the membrane proteins identified in your
> samples?
>
> Best regards,
> Brian
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of William
> Breuer <email obscured>>
> Sent: Tuesday, March 9, 2021 9:36 AM
> To: <email obscured> <email obscured>>
> Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
>
> 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.
>
>
>
> Hi,
> we had reasonable success with the Sulfo-NHS-SS-Biotin cell surface
> labeling kit from Thermo (but I think the kit is actually from Pierce).
> It's called EZ-link-Sulfo-NHS-SS-Biotin, Product # 21331. We ID'd a total
> of 485 proteins, out of which 197 were cell-surface or extracellular
> proteins.
>
> Bill Breuer
> Proteomics/Mass spectrometry Unit,
> Room 1-453, 4th floor, wing 1,
> Institute of Life Sciences,
> Edmond J. Safra Campus - Givat Ram,
> Hebrew University of Jerusalem,
> Jerusalem, 9190401
> Israel
>
> On Tue, Mar 9, 2021 at 1:57 PM Hampton, Brian <email obscured>>
> wrote:
>
> > Hi Yan,
> >
> > I am sorry for not correctly reading your email.  My mistake.
> >
> > I am in the process of trying to analyze the cell membrane proteome of
> two
> > related cell types.  I have come across two different approaches.  One is
> > protease shaving and the other is classic surface biotinylation.
> >
> > The main problem I have with cell surface biotinylation is the large
> > background of cytosolic and nuclear proteins that are nonspecifically
> > captured on the avidin beads.  I'm using Sulfo-NHS-SS-Biotin to label the
> > cells followed by reduction with TCEP to release from the avidin beads.
> > I'm trying different pull-down matrices in hopes of finding one with less
> > non-specific binding.  I'm just using the literature to cobble together a
> > protocol.  One step I want to try next, I have not seen in the
> literature,
> > is to incorporate a ~harshish wash of the beads before release of the
> > biotin bound material.  This could help reduce the non-specific that is
> > recovered following reduction.  I was using a mild detergent initially
> > hoping to also pick up interacting proteins.
> >
> > I did begin the other method of using a protease to shave off proteins
> > exposed on the cell surface.  But it got messy.  I ended up with a fairly
> > large volume and I worried that the method in general would not be
> > reproducible enough to get accurate results for comparisons.
> >
> > If you are looking for a kit, I think Pierce may have this for surface
> > biotin labeling.  I came across a poster of theirs on this topic, but I
> > didn't follow up to check availability.
> >
> > Best regards,
> > Brian
> > ________________________________
> > From: ABRF Discussion Forum <email obscured>> on behalf of Yan Wang <
> > <email obscured>>
> > Sent: Sunday, March 7, 2021 11:21 PM
> > To: ABRF Discussion List <email obscured>>
> > Subject: Re: [ABRF Discussion Forum] membrane protein enrichment
> >
> > 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.
> >
> >
> >
> > Sorry I was not clear. I'm looking for protocols or products for
> enrichment
> > or isolation or cell membrane proteins. Any suggestions?
> > Yan
> >
> > On Sun, Mar 7, 2021 at 1:14 PM Hampton, Brian <
> <email obscured>>
> > wrote:
> >
> > > Hello,
> > >
> > > Do you mean molecular weight cutoff membrane filter concentrators or
> some
> > > form of affinity or charge derivatized membrane for enrichment?  I have
> > > experience with the former but not the latter.  If the latter, I would
> > like
> > > to know what you are considering.
> > >
> > > Thanks,
> > > Brian
> > >
> > > ________________________________
> > > From: ABRF Discussion Forum <email obscured>> on behalf of Yan
> Wang <
> > > <email obscured>>
> > > Sent: Friday, March 5, 2021 6:23 PM
> > > To: ABRF Discussion List <email obscured>>
> > > Subject: [ABRF Discussion Forum] membrane protein enrichment
> > >
> > > 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.
> > >
> > >
> > >
> > > >
> > > > I'm wondering if folks here have good or bad experience with membrane
> > > > protein enrichment products?
> > > >
> > >
> > > Yan
> > >
> > > ――
> > > View topic
> > >
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2Fj2lKnx6hjbX8afQsUHxrU&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999128441%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=V3IrTGCfF385EizajM7D5%2FueuK7xItqqD81h5T1qL5s%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> > > ――
> > > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F1z9BOZpuxXALLkxUV4GS0P&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999128441%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=0A%2F150WO2nqryfEm%2F8LkcKlMRNLS8npYi9eUZOv12vc%3D&amp;reserved=0
> > > Leave group <email obscured>?Subject=Unsubscribe
> > >
> > >
> >
> > ――
> > View topic
> >
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F33lvNigwYjq9uGyBYvQwOY&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=rFHV9IGhKiR1n3HQ0PzPWQB7Ge2At3XaORk7DdEIc1E%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
> > ――
> > View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4bFEJxyuMn3rbCgZCxkE8n&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=jmgblqR9rKltSuPPu%2FDPDm%2F%2BfGcX9MBZebCcoEHrRWc%3D&amp;reserved=0
> > Leave group <email obscured>?Subject=Unsubscribe
> >
> >
>
> ――
> View topic
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F21tFDW3Sw8V7G0ypVTSCBr&amp;data=04%7C01%7Cbhampton%40som.umaryland.edu%7C3289286caadb417103a008d8e308dd97%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637509823999138437%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=vnZvYLxoRwgIqGQmvCYtycjJK0NVEPA5y7Fr3t8tW8E%3D&amp;reserved=0
> Leave group <email obscured>?Subject=Unsubscribe
>
>
> ――
> View topic http://list.abrf.org/r/topic/4b9AtSgevrPLwArjSv15CM
> Leave group <email obscured>?Subject=Unsubscribe
>
>