Hi Hana,
I used the GELFrEE system in the lab in Singapore and was very happy with it,
complex proteomes like from serum worked fine, but also other sources.
Afterwards and for LC-MS/MS AGILENT QTOF with Chip nano HPLC I precipitated the
proteins and removed the SDS with -20 หC EtOH and washed twice with -20 หC
EtOH. Do not try to dry the pellet, keep it - even invisible - slightly wet,
EtOh helps in the digestion. All worked well, the GELFrEE was very good and I
believe, even not tried, is great for membrane proteins. Long time back I
worked with a Uniphor from LKB (very very old school) for Band-3 protein from
red blood cells and this was the only way to separate large fragments. The
GELFrEE should do the same, but much more convenient and samples in parallel.
Manfred
๏ปฟAm 20.08.19, 13:42 schrieb "ABRF Discussion Forum im Auftrag von Hana Koneฤnรก"
<email obscured> im Auftrag von <email obscured>>:
Hi All,
I would like to find out any comments on the Expedeon GELFrEE 8100
fractionation station usage. Especially for protein fractionation before SDS
removal for MS sample prep. Thanks a lot.
Hana
RNDr. Hana Konecna
Central European Institute of Technology CEITEC
Core Facility โ Proteomics
Masaryk University Campus, Building A26
Kamenice 5, 625 00 Brno
Czech Republic
Rest of post
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Hi All,
I would like to find out any comments on the Expedeon GELFrEE 8100
fractionation station usage. Especially for protein fractionation before SDS
removal for MS sample prep. Thanks a lot.
Hana
RNDr. Hana Konecna
Central European Institute of Technology CEITEC
Core Facility โ Proteomics
Masaryk University Campus, Building A26
Kamenice 5, 625 00 Brno
Czech Republic
Hi,
I remember one funny case with similar results. Protein precipitated on top of
the gel and was slowly dissolved by the running buffer and in addition - things
come together - a gap between the gel and the glass plate occurred, maybe due
to the precipitated protein - a very messy gel. But this happened only once
with a self-made gel.
Manfred
๏ปฟAm 13.08.19, 00:31 schrieb "ABRF Discussion Forum im Auftrag von Steve
Hartson" <email obscured> im Auftrag von <email obscured>>:
Hi Y,
I recall a long discussion here several years (decades?) back, regarding
these and related phenom. But I can't find it, so...
In addition to the points made by Brian and Manfred, I recall folks
suggesting (i) proteolysis during lysis (either trace or grotesque), (ii)
"reptation" of proteins through gels or between gels and plates, and (ii)
diffusion of abundant proteins out of the gel and into the staining
solution during the initial staining step (this one is my favorite
explanation: an immunoadsorption gel that also contains lanes loaded with
copious amounts of the "input" lysate always makes me sigh dramatically,
even with several empty intervening lanes). Autosampler carryover is also
a possibility?
The focusing effect of nano-LC and the sensitivity modern mass specs
is...well...just amazing. I frequently see proteins in the "wrong" lanes
or gel segments, esp. if they are abundant. Similarly, high-MW proteins in
low-MW gel regions are pretty familiar too. So I would not necessarily
assume that you're doing anything wrong...
For "protein ID's", I address the dozens - hundreds of proteins ID'd in
"the band" by comparing the peptide/protein intensities of everything
identified in the gel segment; a distribution analysis of these
intensities can greatly set the mind at ease. For shotgun protemics (e.g.,
gel-LC-MS/MS), I concatenate the gel fractions into one sample, so
inter-segment contamination doesn't matter much in my hands.
Cheers and good luck!
S
On Sun, Aug 11, 2019 at 6:40 AM Yishai Levin <email obscured>>
wrote:
> Hi,
> I need some advice regarding shotgun proteomics of reducing gel bands of
> whole cell lysates.
> We often detect similar proteins across bands that migrated relatively
far
> from each other on the gel. Also, in analysis of high mw (>120 kDa)
bands,
> we identify proteins of much lower MW.
> Has anyone experienced this and if so is there a way to improve?
>
> Thanks,
>
> Yishai
>
>
> โโ
> View topic http://list.abrf.org/r/topic/QRoYhjgbJUSMo7J7hJ1Ms
> Leave group <email obscured>?Subject=Unsubscribe
>
>
--
Steven D. Hartson, Ph.D.
Director, Recombinant DNA/Protein Core Facility
Dept. Biochemistry and Molecular Biology
Oklahoma State University
NRC 246
Stillwater, OK 74078-3035
(405)744-6191 phone
(405)744-7799 FAX
http://dnaproteincore.biochem.okstate.edu/
โโ
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Hi Y,
I recall a long discussion here several years (decades?) back, regarding
these and related phenom. But I can't find it, so...
In addition to the points made by Brian and Manfred, I recall folks
suggesting (i) proteolysis during lysis (either trace or grotesque), (ii)
"reptation" of proteins through gels or between gels and plates, and (ii)
diffusion of abundant proteins out of the gel and into the staining
solution during the initial staining step (this one is my favorite
explanation: an immunoadsorption gel that also contains lanes loaded with
copious amounts of the "input" lysate always makes me sigh dramatically,
even with several empty intervening lanes). Autosampler carryover is also
a possibility?
The focusing effect of nano-LC and the sensitivity modern mass specs
is...well...just amazing. I frequently see proteins in the "wrong" lanes
or gel segments, esp. if they are abundant. Similarly, high-MW proteins in
low-MW gel regions are pretty familiar too. So I would not necessarily
assume that you're doing anything wrong...
For "protein ID's", I address the dozens - hundreds of proteins ID'd in
"the band" by comparing the peptide/protein intensities of everything
identified in the gel segment; a distribution analysis of these
intensities can greatly set the mind at ease. For shotgun protemics (e.g.,
gel-LC-MS/MS), I concatenate the gel fractions into one sample, so
inter-segment contamination doesn't matter much in my hands.
Cheers and good luck!
S
On Sun, Aug 11, 2019 at 6:40 AM Yishai Levin <email obscured>>
wrote:
Rest of post
> Hi,
> I need some advice regarding shotgun proteomics of reducing gel bands of
> whole cell lysates.
> We often detect similar proteins across bands that migrated relatively far
> from each other on the gel. Also, in analysis of high mw (>120 kDa) bands,
> we identify proteins of much lower MW.
> Has anyone experienced this and if so is there a way to improve?
>
> Thanks,
>
> Yishai
>
>
> โโ
> View topic http://list.abrf.org/r/topic/QRoYhjgbJUSMo7J7hJ1Ms
> Leave group <email obscured>?Subject=Unsubscribe
>
>
--
Steven D. Hartson, Ph.D.
Director, Recombinant DNA/Protein Core Facility
Dept. Biochemistry and Molecular Biology
Oklahoma State University
NRC 246
Stillwater, OK 74078-3035
(405)744-6191 phone
(405)744-7799 FAX
http://dnaproteincore.biochem.okstate.edu/
Hello Yishai,
I presume you refer to within the same lane when you remark about detecting
similar proteins across bands that migrated far from each other. If so then I
think this is due to aggregation and smearing during electrophoresis. You
could try dissolving dry urea to the sample in sample buffer directly before
running the gel to make it 6 - 8 molar. You will increase the original volume
of the sample by about 2-fold. Do not heat urea or you will generate
cyanate.... much more quickly.
Recently I noticed that a very large MW receptor (detected via western blot)
became or remained significantly aggregated when extracted using 5% SDS then
diluted into 2x Laemmli sample buffer for the gel. However, the very same
sample extracted in 1% dodecylmaltoside then diluted into sample buffer for the
gel showed no aggregation. This aggregation in the SDS extracted sample
manifested itself as reduced intensity of the band at the expected MW of the
protein (compared to the DDM extracted sample) and smearing up to the bottom of
the well with fairly bright intensity at the bottom the well in the western
blot. The DDM extracted sample had none of this.
Point being that if one were to do proteomics on a band in that smeared region
one would probably detect this protein at any higher MW than predicted.
There is this (well founded) notion that SDS is the universal solubilizer and
the notion that proteins behave uniformly in its presence. That is the case
for many proteins but probably not all and further disruption of structure
following reduction may exacerbate solubility for some as the proteins run
through the gel. Consider that the micro-environment of the proteins changes
from when it is in sample buffer to as it runs through the gel. The first
change is a massive increase in protein concentration as it is compressed by
the ion front and electrical field to the bottom of the well and into the
stacking gel. I imagine not all proteins maintain full solubility at this high
protein concentration and the smearing begins.
Also, if you are only reducing and not alkylating disulfide bonds prior to
running the gel then as the proteins run into the gel, they leave behind the
reducing agent (e.g. DTT) and then disulfide scrambling could occur. For
example in the old days of 2D gels it was always recommended to reduce and
alkylate the first dimension IEF strip to prevent vertical streaking in the
second dimension separation SDS gel. The vertical streaking would cause
certain proteins to be present across the gel vertically.
Let us know if you succeed in your troubleshooting efforts.
Best of luck.
Regards,
Rest of post
Brian
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Yishai Levin
<email obscured>>
Sent: Sunday, August 11, 2019 7:40 AM
To: <email obscured> <email obscured>>
Subject: [ABRF Discussion Forum] Gel bands
Hi,
I need some advice regarding shotgun proteomics of reducing gel bands of whole
cell lysates.
We often detect similar proteins across bands that migrated relatively far from
each other on the gel. Also, in analysis of high mw (>120 kDa) bands, we
identify proteins of much lower MW.
Has anyone experienced this and if so is there a way to improve?
Thanks,
Yishai
โโ
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Hey Yishai,
sounds strange, do you reduce and alkylate the proteins before separation, do
you use home-made or commercial gels? On you Coomassie stained gel, do you see
band or smear - maybe overloaded or sample run into the neighboring pockets, do
you leave one lane between samples free to avoid cross-contamination? I have
seen this only in a few cases, overloading, cross contamination. And how is the
sequence coverage of the proteins, low MW and high MW.
Need some more information to help
Manfred
๏ปฟAm 11.08.19, 13:40 schrieb "ABRF Discussion Forum im Auftrag von Yishai Levin"
<email obscured> im Auftrag von <email obscured>>:
Hi,
I need some advice regarding shotgun proteomics of reducing gel bands of
whole cell lysates.
We often detect similar proteins across bands that migrated relatively far
from each other on the gel. Also, in analysis of high mw (>120 kDa) bands, we
identify proteins of much lower MW.
Has anyone experienced this and if so is there a way to improve?
Thanks,
Yishai
โโ
View topic http://list.abrf.org/r/topic/QRoYhjgbJUSMo7J7hJ1Ms
Leave group <email obscured>?Subject=Unsubscribe
Hi,
I need some advice regarding shotgun proteomics of reducing gel bands of whole
cell lysates.
We often detect similar proteins across bands that migrated relatively far from
each other on the gel. Also, in analysis of high mw (>120 kDa) bands, we
identify proteins of much lower MW.
Has anyone experienced this and if so is there a way to improve?
The Proteomics Center at University of Missouri, Columbia, MO, would probably
be able to help you.
Contact either Dr. Brian Mooney at 573-884-7374 or Dr. Michael Greenlief at
573-882-3288.
B. DaGue
retiree (from the above lab)
Rest of post
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Peter Felker
<email obscured>>
Sent: Thursday, August 1, 2019 2:33 PM
To: <email obscured>
Subject: [ABRF Discussion Forum] Suggestions for cost effective, small volume,
small molecule mass spec services
Hello
Every 6-8 months we need to have several unknown collected peaks from HPLC
ZIC HILIC column analysed and hopefully some structures proposed.
We would appreciate suggestions on who to go for these services
Thank you
Peter
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Ding, Hua
<email obscured>>
Sent: Thursday, August 1, 2019 3:27 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Thank you for sharing!
We use artificially acetylated lysozyme as internal standard.
The enrichment specificity is really sample dependent.
Best,
Hua
Hua Ding, Ph.D.
Research Associate
Protein and Proteomics Core Facility
The Children's Hospital of Philadelphia
<email obscured>
Phone: 267-426-5552
-----Original Message-----
From: ABRF Discussion Forum <email obscured>] On Behalf Of J. Will
Thompson, Ph.D.
Sent: Wednesday, July 31, 2019 8:27 AM
To: <email obscured>
Subject: [External] Re: [ABRF Discussion Forum] IP of lysine acetylated
peptides
Markus
The enrichment specificity seems to vary pretty widely based on the starting
matrix. From cell lines which are highly enriched in acetylation, we might see
as much as 50-60% specificity. However, we more often see 20-25% specificity
for samples such as tissue. I have done dedicated recovery experiments with
stable-isotope labeled acetylated peptides, and the absolute recovery varies
extremely widely between peptides. The pan-acetyl antibodies definitely have
varying binding strengths for different background sequences, and I think this
likely has to do with whatever acetylated sequences were used to produce the
antibodies. (We realized the variability in recovery in the context of the PRM
experiment described in
https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29531224&data=02%7C01%7C%7C77a2e19bf3db40fa7f1608d71694c465%7C84df9e7fe9f640afb435aaaaaaaaaaaa%7C1%7C0%7C637002700509638048&sdata=xB8Tcoz6LHP4sf9g0YnVNMhs7Lmd7hncN0g60glKDf0%3D&reserved=0).
Another thing we have done which has been extremely helpful with respect to
determining consistency between pulldowns is to use a protein-level spike of
artificially acetylated BSA, added at about 6 parts per million prior to the
sample digestion
(https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F26748706&data=02%7C01%7C%7C77a2e19bf3db40fa7f1608d71694c465%7C84df9e7fe9f640afb435aaaaaaaaaaaa%7C1%7C0%7C637002700509648035&sdata=kTXHTdLQg2nS%2FHvSnLtTqFmlDMRBrAyKiuQKt3yO4lk%3D&reserved=0).
NHS-acetylated BSA will generate about 40 Kac peptides which can serve as
control peptides between your samples for normalization and to help set
quantitative cutoffs for fold-changes and differential expression.
Cheers
Will
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of markus.hartl
Sent: Wednesday, July 31, 2019 4:35 AM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Hi everyone,
thanks for this interesting thread!
We have also tried the Cell Signaling antibody and it worked very well for us.
We basically followed the protocol without any major changes. I totally agree
that you will need a few mg to start with if you want to get high coverage. We
also used a digest in 8M urea and lyophilized as described by Will.
The observation about the urea digest is interesting. We successfully used FASP
for mapping lysine acetylation in Arabidopsis in the past - this was the only
protocol that seemed to work for plant material. FASP at this scale is not much
fun to do, so I was happy to move back to urea digests for mammalian cells.
Before the new CST antibody became available we successfully used the
Immunechem antibody. However, the batch-to-batch variation could be huge and we
always tested the lot with a standard sample before ordering larger amounts to
make sure it works well. I have heard similar rumours about CST antibody
batch-to-batch variation but so far cannot confirm them (only had one batch
which worked great). Would be great if Kim could comment on this.
Will, could you comment on the enrichment efficiency that you obtained? We were
in the range of 50-60% and I wonder if that could be improved somehow.
One final remark: do not be disappointed when you look for the first time at
the TIC chromatogram because at first glance it might seem as if nothing has
worked. The huge histone signals dominate the chromatogram and make the other
analytes often seem like background.
Cheers,
Markus Hartl
Max Perutz Labs Vienna, Mass Spectrometry Facility
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Hello
Every 6-8 months we need to have several unknown collected peaks from HPLC
ZIC HILIC column analysed and hopefully some structures proposed.
We would appreciate suggestions on who to go for these services
Thank you
Rest of post
Peter
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Ding, Hua
<email obscured>>
Sent: Thursday, August 1, 2019 3:27 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Thank you for sharing!
We use artificially acetylated lysozyme as internal standard.
The enrichment specificity is really sample dependent.
Best,
Hua
Hua Ding, Ph.D.
Research Associate
Protein and Proteomics Core Facility
The Children's Hospital of Philadelphia
<email obscured>
Phone: 267-426-5552
-----Original Message-----
From: ABRF Discussion Forum <email obscured>] On Behalf Of J. Will
Thompson, Ph.D.
Sent: Wednesday, July 31, 2019 8:27 AM
To: <email obscured>
Subject: [External] Re: [ABRF Discussion Forum] IP of lysine acetylated
peptides
Markus
The enrichment specificity seems to vary pretty widely based on the starting
matrix. From cell lines which are highly enriched in acetylation, we might see
as much as 50-60% specificity. However, we more often see 20-25% specificity
for samples such as tissue. I have done dedicated recovery experiments with
stable-isotope labeled acetylated peptides, and the absolute recovery varies
extremely widely between peptides. The pan-acetyl antibodies definitely have
varying binding strengths for different background sequences, and I think this
likely has to do with whatever acetylated sequences were used to produce the
antibodies. (We realized the variability in recovery in the context of the PRM
experiment described in
https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29531224&data=02%7C01%7C%7C77a2e19bf3db40fa7f1608d71694c465%7C84df9e7fe9f640afb435aaaaaaaaaaaa%7C1%7C0%7C637002700509638048&sdata=xB8Tcoz6LHP4sf9g0YnVNMhs7Lmd7hncN0g60glKDf0%3D&reserved=0).
Another thing we have done which has been extremely helpful with respect to
determining consistency between pulldowns is to use a protein-level spike of
artificially acetylated BSA, added at about 6 parts per million prior to the
sample digestion
(https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F26748706&data=02%7C01%7C%7C77a2e19bf3db40fa7f1608d71694c465%7C84df9e7fe9f640afb435aaaaaaaaaaaa%7C1%7C0%7C637002700509648035&sdata=kTXHTdLQg2nS%2FHvSnLtTqFmlDMRBrAyKiuQKt3yO4lk%3D&reserved=0).
NHS-acetylated BSA will generate about 40 Kac peptides which can serve as
control peptides between your samples for normalization and to help set
quantitative cutoffs for fold-changes and differential expression.
Cheers
Will
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of markus.hartl
Sent: Wednesday, July 31, 2019 4:35 AM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Hi everyone,
thanks for this interesting thread!
We have also tried the Cell Signaling antibody and it worked very well for us.
We basically followed the protocol without any major changes. I totally agree
that you will need a few mg to start with if you want to get high coverage. We
also used a digest in 8M urea and lyophilized as described by Will.
The observation about the urea digest is interesting. We successfully used FASP
for mapping lysine acetylation in Arabidopsis in the past - this was the only
protocol that seemed to work for plant material. FASP at this scale is not much
fun to do, so I was happy to move back to urea digests for mammalian cells.
Before the new CST antibody became available we successfully used the
Immunechem antibody. However, the batch-to-batch variation could be huge and we
always tested the lot with a standard sample before ordering larger amounts to
make sure it works well. I have heard similar rumours about CST antibody
batch-to-batch variation but so far cannot confirm them (only had one batch
which worked great). Would be great if Kim could comment on this.
Will, could you comment on the enrichment efficiency that you obtained? We were
in the range of 50-60% and I wonder if that could be improved somehow.
One final remark: do not be disappointed when you look for the first time at
the TIC chromatogram because at first glance it might seem as if nothing has
worked. The huge histone signals dominate the chromatogram and make the other
analytes often seem like background.
Cheers,
Markus Hartl
Max Perutz Labs Vienna, Mass Spectrometry Facility
โโ
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Thank you for sharing!
We use artificially acetylated lysozyme as internal standard.
The enrichment specificity is really sample dependent.
Best,
Hua
Hua Ding, Ph.D.
Research Associate
Protein and Proteomics Core Facility
The Children's Hospital of Philadelphia
<email obscured>
Phone: 267-426-5552
Rest of post
-----Original Message-----
From: ABRF Discussion Forum <email obscured>] On Behalf Of J. Will
Thompson, Ph.D.
Sent: Wednesday, July 31, 2019 8:27 AM
To: <email obscured>
Subject: [External] Re: [ABRF Discussion Forum] IP of lysine acetylated
peptides
Markus
The enrichment specificity seems to vary pretty widely based on the starting
matrix. From cell lines which are highly enriched in acetylation, we might see
as much as 50-60% specificity. However, we more often see 20-25% specificity
for samples such as tissue. I have done dedicated recovery experiments with
stable-isotope labeled acetylated peptides, and the absolute recovery varies
extremely widely between peptides. The pan-acetyl antibodies definitely have
varying binding strengths for different background sequences, and I think this
likely has to do with whatever acetylated sequences were used to produce the
antibodies. (We realized the variability in recovery in the context of the PRM
experiment described in https://www.ncbi.nlm.nih.gov/pubmed/29531224).
Another thing we have done which has been extremely helpful with respect to
determining consistency between pulldowns is to use a protein-level spike of
artificially acetylated BSA, added at about 6 parts per million prior to the
sample digestion (https://www.ncbi.nlm.nih.gov/pubmed/26748706).
NHS-acetylated BSA will generate about 40 Kac peptides which can serve as
control peptides between your samples for normalization and to help set
quantitative cutoffs for fold-changes and differential expression.
Cheers
Will
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of markus.hartl
Sent: Wednesday, July 31, 2019 4:35 AM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Hi everyone,
thanks for this interesting thread!
We have also tried the Cell Signaling antibody and it worked very well for us.
We basically followed the protocol without any major changes. I totally agree
that you will need a few mg to start with if you want to get high coverage. We
also used a digest in 8M urea and lyophilized as described by Will.
The observation about the urea digest is interesting. We successfully used FASP
for mapping lysine acetylation in Arabidopsis in the past - this was the only
protocol that seemed to work for plant material. FASP at this scale is not much
fun to do, so I was happy to move back to urea digests for mammalian cells.
Before the new CST antibody became available we successfully used the
Immunechem antibody. However, the batch-to-batch variation could be huge and we
always tested the lot with a standard sample before ordering larger amounts to
make sure it works well. I have heard similar rumours about CST antibody
batch-to-batch variation but so far cannot confirm them (only had one batch
which worked great). Would be great if Kim could comment on this.
Will, could you comment on the enrichment efficiency that you obtained? We were
in the range of 50-60% and I wonder if that could be improved somehow.
One final remark: do not be disappointed when you look for the first time at
the TIC chromatogram because at first glance it might seem as if nothing has
worked. The huge histone signals dominate the chromatogram and make the other
analytes often seem like background.
Cheers,
Markus Hartl
Max Perutz Labs Vienna, Mass Spectrometry Facility
โโ
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Thanks to everyone sharing your information here, this is extremely helpful
especially as we are also starting to venture into a new acetylation project!
Will and Kim, thanks for your comments on specificity, it seems I have been
very lucky with the cells and tissues that I have been looking at so far. Will,
I like your BSA spike-in idea very much, I will definitely consider this for QC
in the future.
Hi Markus, Duncan, and all,
Thanks for your insights and comments. Regarding lot to lot variation, all
the CST PTMScan kit antibodies are not only monoclonal, but are expressed
recombinantly, so the lot variation is quite minimal. Nevertheless, each
new lot of bead-bound antibody is tested side by side against the previous
lot prior to release, by immunoenrichment of mouse liver tryptic peptides
(in the case of K-Ac) or another appropriate standard sample. We do
occasionally hear from customers who suspect a problem with a new lot, and
we're happy to work together to troubleshoot, since we obviously want to be
aware and correct any problems as soon as possible. The outcome of this
work usually ends up identifying a different variable as the culprit
however.
I just want to add my 2 cents about your enrichment efficiency - as Will
said, 50% enrichment using an antibody-based method is excellent. We would
consider that a great success, and lower percentages of target peptides in
the final sample, particularly for lower-level modifications like
phosphotyrosine, would also be expected. MS instruments are so sensitive
now that the background level of unmodified peptides that stick to the
beads and Protein A are easily detectable, so particularly for samples with
lower levels of modified peptides (or if you use lower amounts of starting
peptide), many unmodified peptides will be detected along with the many
modified peptides.
One final note particularly for K-Ac peptide enrichment, we've recently
changed our recommended protocol to performing the protein reduction at
room temperature for 60 minutes rather 55 degrees. This change is to
reduce the level of carbamylation in the sample due to heating the urea, as
the K-Ac antibody can recognize carbamylated lysines as well. This finding
was published by Javier Munoz's lab a couple years ago in a really nice
study, and we've confirmed it internally. We find reducing at room temp
results in greatly reduced carbamylation and improved K-Ac peptide
recovery.
Duncan, yes, Chuck's passport is active =), so we would be happy to discuss
scheduling training at your site - I'll contact you off line.
Rest of post
Best,
Kim
--
Kimberly A. Lee, Ph.D.
Director, Proteomics
Cell Signaling Technology
3 Trask Lane
Danvers, MA 01923
Office: 978-624-3205
Cell: 978-412-2300 <(978)%20412-2300>
<email obscured>
www.cellsignal.com/proteomics
On Wed, Jul 31, 2019 at 8:26 AM J. Will Thompson, Ph.D. <
<email obscured>> wrote:
> Markus
>
> The enrichment specificity seems to vary pretty widely based on the
> starting matrix. From cell lines which are highly enriched in acetylation,
> we might see as much as 50-60% specificity. However, we more often see
> 20-25% specificity for samples such as tissue. I have done dedicated
> recovery experiments with stable-isotope labeled acetylated peptides, and
> the absolute recovery varies extremely widely between peptides. The
> pan-acetyl antibodies definitely have varying binding strengths for
> different background sequences, and I think this likely has to do with
> whatever acetylated sequences were used to produce the antibodies. (We
> realized the variability in recovery in the context of the PRM experiment
> described in https://www.ncbi.nlm.nih.gov/pubmed/29531224).
>
> Another thing we have done which has been extremely helpful with respect
> to determining consistency between pulldowns is to use a protein-level
> spike of artificially acetylated BSA, added at about 6 parts per million
> prior to the sample digestion (
> https://www.ncbi.nlm.nih.gov/pubmed/26748706). NHS-acetylated BSA will
> generate about 40 Kac peptides which can serve as control peptides between
> your samples for normalization and to help set quantitative cutoffs for
> fold-changes and differential expression.
>
> Cheers
>
> Will
>
> -----Original Message-----
> From: ABRF Discussion Forum <email obscured>> On Behalf Of markus.hartl
> Sent: Wednesday, July 31, 2019 4:35 AM
> To: <email obscured>
> Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
>
> Hi everyone,
>
> thanks for this interesting thread!
>
> We have also tried the Cell Signaling antibody and it worked very well for
> us. We basically followed the protocol without any major changes. I totally
> agree that you will need a few mg to start with if you want to get high
> coverage. We also used a digest in 8M urea and lyophilized as described by
> Will.
>
> The observation about the urea digest is interesting. We successfully used
> FASP for mapping lysine acetylation in Arabidopsis in the past - this was
> the only protocol that seemed to work for plant material. FASP at this
> scale is not much fun to do, so I was happy to move back to urea digests
> for mammalian cells.
>
> Before the new CST antibody became available we successfully used the
> Immunechem antibody. However, the batch-to-batch variation could be huge
> and we always tested the lot with a standard sample before ordering larger
> amounts to make sure it works well. I have heard similar rumours about CST
> antibody batch-to-batch variation but so far cannot confirm them (only had
> one batch which worked great). Would be great if Kim could comment on this.
>
> Will, could you comment on the enrichment efficiency that you obtained? We
> were in the range of 50-60% and I wonder if that could be improved somehow.
>
> One final remark: do not be disappointed when you look for the first time
> at the TIC chromatogram because at first glance it might seem as if nothing
> has worked. The huge histone signals dominate the chromatogram and make the
> other analytes often seem like background.
>
> Cheers,
>
> Markus Hartl
> Max Perutz Labs Vienna, Mass Spectrometry Facility
>
>
>
> โโ
> View topic
>
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>
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Hello Guys
The ABRF discussion forum and its wonderful people never disappoint!
Thanks so much to Brian, Will, Kim, Jon and Markus for there guidance and
experience.
I'm now off into the Acetylome with a far greater appreciation :-)
Kim, do you think we could get some CST guidance & assistance on this project
her in Manchester (the very ununited kingdom). Jon is also interested so we may
have a cluster of interest in the state of Manchuria.
Markus
The enrichment specificity seems to vary pretty widely based on the starting
matrix. From cell lines which are highly enriched in acetylation, we might see
as much as 50-60% specificity. However, we more often see 20-25% specificity
for samples such as tissue. I have done dedicated recovery experiments with
stable-isotope labeled acetylated peptides, and the absolute recovery varies
extremely widely between peptides. The pan-acetyl antibodies definitely have
varying binding strengths for different background sequences, and I think this
likely has to do with whatever acetylated sequences were used to produce the
antibodies. (We realized the variability in recovery in the context of the PRM
experiment described in https://www.ncbi.nlm.nih.gov/pubmed/29531224).
Another thing we have done which has been extremely helpful with respect to
determining consistency between pulldowns is to use a protein-level spike of
artificially acetylated BSA, added at about 6 parts per million prior to the
sample digestion (https://www.ncbi.nlm.nih.gov/pubmed/26748706).
NHS-acetylated BSA will generate about 40 Kac peptides which can serve as
control peptides between your samples for normalization and to help set
quantitative cutoffs for fold-changes and differential expression.
Rest of post
Cheers
Will
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of markus.hartl
Sent: Wednesday, July 31, 2019 4:35 AM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Hi everyone,
thanks for this interesting thread!
We have also tried the Cell Signaling antibody and it worked very well for us.
We basically followed the protocol without any major changes. I totally agree
that you will need a few mg to start with if you want to get high coverage. We
also used a digest in 8M urea and lyophilized as described by Will.
The observation about the urea digest is interesting. We successfully used FASP
for mapping lysine acetylation in Arabidopsis in the past - this was the only
protocol that seemed to work for plant material. FASP at this scale is not much
fun to do, so I was happy to move back to urea digests for mammalian cells.
Before the new CST antibody became available we successfully used the
Immunechem antibody. However, the batch-to-batch variation could be huge and we
always tested the lot with a standard sample before ordering larger amounts to
make sure it works well. I have heard similar rumours about CST antibody
batch-to-batch variation but so far cannot confirm them (only had one batch
which worked great). Would be great if Kim could comment on this.
Will, could you comment on the enrichment efficiency that you obtained? We were
in the range of 50-60% and I wonder if that could be improved somehow.
One final remark: do not be disappointed when you look for the first time at
the TIC chromatogram because at first glance it might seem as if nothing has
worked. The huge histone signals dominate the chromatogram and make the other
analytes often seem like background.
Cheers,
Markus Hartl
Max Perutz Labs Vienna, Mass Spectrometry Facility
โโ
View topic
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Hi everyone,
thanks for this interesting thread!
We have also tried the Cell Signaling antibody and it worked very well for us.
We basically followed the protocol without any major changes. I totally agree
that you will need a few mg to start with if you want to get high coverage. We
also used a digest in 8M urea and lyophilized as described by Will.
The observation about the urea digest is interesting. We successfully used FASP
for mapping lysine acetylation in Arabidopsis in the past - this was the only
protocol that seemed to work for plant material. FASP at this scale is not much
fun to do, so I was happy to move back to urea digests for mammalian cells.
Before the new CST antibody became available we successfully used the
Immunechem antibody. However, the batch-to-batch variation could be huge and we
always tested the lot with a standard sample before ordering larger amounts to
make sure it works well. I have heard similar rumours about CST antibody
batch-to-batch variation but so far cannot confirm them (only had one batch
which worked great). Would be great if Kim could comment on this.
Will, could you comment on the enrichment efficiency that you obtained? We were
in the range of 50-60% and I wonder if that could be improved somehow.
One final remark: do not be disappointed when you look for the first time at
the TIC chromatogram because at first glance it might seem as if nothing has
worked. The huge histone signals dominate the chromatogram and make the other
analytes often seem like background.
Cheers,
Markus Hartl
Max Perutz Labs Vienna, Mass Spectrometry Facility
Hi Duncan,
Very interesting discussion so thanks for everyone for contributing.
If you were to set up any on-site training near Mcr we'd be interested in
attending and making the numbers up :-).
Jon
Hi Duncan and all,
I just wanted to mention that CST is happy to provide advice and support to
new (and experienced) users of the PTMScan peptide IP kits. As Will
mentioned, we can also provide on-site hands-on training for a group of
interested scientists. Chuck Farnsworth <email obscured>) is
the contact person for any labs interested in hosting a training session -
feel free to reach out to him, or to me.
Regarding the need for mg-level samples, I would agree with all the
comments so far. I've attached a graph of our data looking at number of
identified unique Kac-containing peptides as a function of input sample
amount, using a mouse liver peptide sample and the Kac peptide enrichment
kit. The data were acquired on our Lumos using a 90 minute gradient. The
mouse liver sample is pretty rich in Kac, so if the starting sample had a
lower level of modification, we would expect the impact of reduction in
starting material to be even more significant.
[image: image.png]
Best,
Rest of post
Kim
--
Kimberly A. Lee, Ph.D.
Director, Proteomics
Cell Signaling Technology
3 Trask Lane
Danvers, MA 01923
Office: 978-624-3205
Cell: 978-412-2300 <(978)%20412-2300>
<email obscured>
www.cellsignal.com/proteomics
On Tue, Jul 30, 2019 at 3:42 PM J. Will Thompson, Ph.D. <
<email obscured>> wrote:
> Hi Duncan
>
> We have used CST Ab pulldowns for Kac and other PTM enrichments for a
> number of years with good success. We have found several key aspects, some
> of which Brian alluded to:
>
> 1. Starting material. For antibody enrichments, starting with less than 5
> mg almost always has led to only limited success. The lower level the PTM,
> the higher amount needed.
>
> 2. Digestion procedure. We have had limited success with any digestion
> procedure other than 8M Urea, trypsin, followed by C18 SPE. I have not
> ever been very successful deviating from this digestion approach for the
> purposes of the pulldown, even though we use a wide variety of digestion
> approaches for general proteomics projects. I know this seems ridiculous,
> but it has been my observation.
>
> 3. Lyophilization. For whatever reason, and I also have not ever been
> able to explain this, but freeze-drying (lyophilizing) the peptides after
> digestion and SPE seems to be critical. Speedvac dried samples just do not
> seem to work in the IP. I think this has something to do with the
> lyophilized peptides coming back into solution better and getting rid of
> the acid in the samples, but this also seems to be key.
>
> These are the key points we have observed. Our lab has successfully
> performed experiments with anti-K(GG), anti-Kac, anti-pY, and a host of
> phos motif antibodies from CST, essentially with the same digestion and
> enrichment protocol. I will say that we received hands-on training from
> CST scientists when we first purchased the kits and that was very helpful;
> we have deviated only in small ways from those original protocols (to
> improve purity by washing with different buffers after IP, for instance).
>
> Cheers
>
> Will Thompson
> Duke Proteomics and Metabolomics Shared Resource
>
> -----Original Message-----
> From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton,
> Brian
> Sent: Tuesday, July 30, 2019 3:08 PM
> To: <email obscured>
> Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
>
> Hi Duncan,
>
> A lab here is using the Cell Signaling Ab kit for AcK. They had done this
> on a subcellular fraction from a particular anatomical part of a single
> mouse brain and the success was definitely lacking. I attribute this to
> the low amount of starting material which was less than ~200ug of protein
> extract. I generated the peptides using my own protocol which works well
> on that amount of protein as starting material. Then handed the peptide
> prep back to the research lab to do the AcK pull down. The limited amount
> of peptides IDโd showed the expected proteins but didnโt produce a deep
> enough data set to be useful. They are going to scale up.
>
> So it seems that a crucial aspect is a large amount of protein as starting
> material. In the literature they are using multi milligram amounts of
> protein extract and having success. We used microgram amounts and had very
> limited success.
>
> Birgit Schilling at the Buck Institute has used the Cell Signaling Ab in a
> recent publication with success using milligram amounts of protein extract.
>
https://urldefense.proofpoint.com/v2/url?u=https-3A__dx.doi.org_10.1002-252Fpmic.201800123&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=ck3NgrHt92AGyLd3Lv8_IYJHZLYVpM1L45uwMnL_q-U&s=pHmLj1CAh9f0ZmQlEBOKPXFr39URELYpjw8l3JAtDgk&e=
> <
>
https://urldefense.proofpoint.com/v2/url?u=https-3A__dx.doi.org_10.1002_pmic.201800123&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=ck3NgrHt92AGyLd3Lv8_IYJHZLYVpM1L45uwMnL_q-U&s=WCrNuSOT_nSl5yit-_3tNdjQN6b2qlA5hhHxbrhVZwk&e=
> >
>
>
> (Off question response) Non Ab approach:
>
> Another approach may work uses N-acetoxy-[2H3]succinimide to label all
> unmodified lysine. After labeling the extract you have heavy AcK
> representing originally unmodified K and light AcK which represents what
> the cell modified. So its differential isotopic labeling. See: โLysine
> acetylation stoichiometry and proteomics analyses reveal pathways regulated
> by sirtuin 1 in human cellsโ
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.jbc.org_content_292_44_18129&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=ck3NgrHt92AGyLd3Lv8_IYJHZLYVpM1L45uwMnL_q-U&s=cRHump1Fj4KrBE2gBtDnsh9ruOaHzw_B9UhyFozCTLM&e=
> . You have to make the deuterated NAS but it is a straight forward
> reaction.
>
>
> Regards,
>
> Brian
>
> On Jul 30, 2019, at 2:31 PM, duncan.smith <email obscured>
> <email obscured>>> wrote:
>
> Hello there
>
> Is anyone having success at profiling lysine acetylated peptides using an
> immunoprecipitation enrichment strategy? Would anyone be willing to share
> their experience of which commercially available antibodies and IP
> approaches work well (or indeed do not work well) in their hands? The
> literature is the literature but direct comments on these types of
> approaches are always more useful in my opinion :-)
>
> Many thanks in advance for help
>
> Duncan
> โโ
> View topic
>
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protected by attorney-client privilege. ย Unless you are the addressee, you
may not use, copy or disclose to anyone this message or any information
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Hi Duncan
We have used CST Ab pulldowns for Kac and other PTM enrichments for a number of
years with good success. We have found several key aspects, some of which
Brian alluded to:
1. Starting material. For antibody enrichments, starting with less than 5 mg
almost always has led to only limited success. The lower level the PTM, the
higher amount needed.
2. Digestion procedure. We have had limited success with any digestion
procedure other than 8M Urea, trypsin, followed by C18 SPE. I have not ever
been very successful deviating from this digestion approach for the purposes of
the pulldown, even though we use a wide variety of digestion approaches for
general proteomics projects. I know this seems ridiculous, but it has been my
observation.
3. Lyophilization. For whatever reason, and I also have not ever been able to
explain this, but freeze-drying (lyophilizing) the peptides after digestion and
SPE seems to be critical. Speedvac dried samples just do not seem to work in
the IP. I think this has something to do with the lyophilized peptides coming
back into solution better and getting rid of the acid in the samples, but this
also seems to be key.
These are the key points we have observed. Our lab has successfully performed
experiments with anti-K(GG), anti-Kac, anti-pY, and a host of phos motif
antibodies from CST, essentially with the same digestion and enrichment
protocol. I will say that we received hands-on training from CST scientists
when we first purchased the kits and that was very helpful; we have deviated
only in small ways from those original protocols (to improve purity by washing
with different buffers after IP, for instance).
Cheers
Will Thompson
Duke Proteomics and Metabolomics Shared Resource
Rest of post
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian
Sent: Tuesday, July 30, 2019 3:08 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] IP of lysine acetylated peptides
Hi Duncan,
A lab here is using the Cell Signaling Ab kit for AcK. They had done this on a
subcellular fraction from a particular anatomical part of a single mouse brain
and the success was definitely lacking. I attribute this to the low amount of
starting material which was less than ~200ug of protein extract. I generated
the peptides using my own protocol which works well on that amount of protein
as starting material. Then handed the peptide prep back to the research lab to
do the AcK pull down. The limited amount of peptides IDโd showed the expected
proteins but didnโt produce a deep enough data set to be useful. They are
going to scale up.
So it seems that a crucial aspect is a large amount of protein as starting
material. In the literature they are using multi milligram amounts of protein
extract and having success. We used microgram amounts and had very limited
success.
Birgit Schilling at the Buck Institute has used the Cell Signaling Ab in a
recent publication with success using milligram amounts of protein extract.
https://urldefense.proofpoint.com/v2/url?u=https-3A__dx.doi.org_10.1002-252Fpmic.201800123&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=ck3NgrHt92AGyLd3Lv8_IYJHZLYVpM1L45uwMnL_q-U&s=pHmLj1CAh9f0ZmQlEBOKPXFr39URELYpjw8l3JAtDgk&e=
<https://urldefense.proofpoint.com/v2/url?u=https-3A__dx.doi.org_10.1002_pmic.201800123&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=ck3NgrHt92AGyLd3Lv8_IYJHZLYVpM1L45uwMnL_q-U&s=WCrNuSOT_nSl5yit-_3tNdjQN6b2qlA5hhHxbrhVZwk&e=
>
(Off question response) Non Ab approach:
Another approach may work uses N-acetoxy-[2H3]succinimide to label all
unmodified lysine. After labeling the extract you have heavy AcK representing
originally unmodified K and light AcK which represents what the cell modified.
So its differential isotopic labeling. See: โLysine acetylation stoichiometry
and proteomics analyses reveal pathways regulated by sirtuin 1 in human cellsโ
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.jbc.org_content_292_44_18129&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=ck3NgrHt92AGyLd3Lv8_IYJHZLYVpM1L45uwMnL_q-U&s=cRHump1Fj4KrBE2gBtDnsh9ruOaHzw_B9UhyFozCTLM&e=
. You have to make the deuterated NAS but it is a straight forward reaction.
Regards,
Brian
On Jul 30, 2019, at 2:31 PM, duncan.smith
<email obscured><email obscured>>>
wrote:
Hello there
Is anyone having success at profiling lysine acetylated peptides using an
immunoprecipitation enrichment strategy? Would anyone be willing to share their
experience of which commercially available antibodies and IP approaches work
well (or indeed do not work well) in their hands? The literature is the
literature but direct comments on these types of approaches are always more
useful in my opinion :-)
Many thanks in advance for help
Duncan
โโ
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Hi Duncan,
A lab here is using the Cell Signaling Ab kit for AcK. They had done this on a
subcellular fraction from a particular anatomical part of a single mouse brain
and the success was definitely lacking. I attribute this to the low amount of
starting material which was less than ~200ug of protein extract. I generated
the peptides using my own protocol which works well on that amount of protein
as starting material. Then handed the peptide prep back to the research lab to
do the AcK pull down. The limited amount of peptides IDโd showed the expected
proteins but didnโt produce a deep enough data set to be useful. They are
going to scale up.
So it seems that a crucial aspect is a large amount of protein as starting
material. In the literature they are using multi milligram amounts of protein
extract and having success. We used microgram amounts and had very limited
success.
Birgit Schilling at the Buck Institute has used the Cell Signaling Ab in a
recent publication with success using milligram amounts of protein extract.
https://dx.doi.org/10.1002%2Fpmic.201800123<https://dx.doi.org/10.1002/pmic.201800123>
(Off question response) Non Ab approach:
Another approach may work uses N-acetoxy-[2H3]succinimide to label all
unmodified lysine. After labeling the extract you have heavy AcK representing
originally unmodified K and light AcK which represents what the cell modified.
So its differential isotopic labeling. See: โLysine acetylation stoichiometry
and proteomics analyses reveal pathways regulated by sirtuin 1 in human cellsโ
http://www.jbc.org/content/292/44/18129. You have to make the deuterated NAS
but it is a straight forward reaction.
Regards,
Brian
On Jul 30, 2019, at 2:31 PM, duncan.smith
<email obscured><email obscured>>>
wrote:
Hello there
Is anyone having success at profiling lysine acetylated peptides using an
immunoprecipitation enrichment strategy? Would anyone be willing to share their
experience of which commercially available antibodies and IP approaches work
well (or indeed do not work well) in their hands? The literature is the
literature but direct comments on these types of approaches are always more
useful in my opinion :-)
Many thanks in advance for help
Duncan
โโ
View topic http://list.abrf.org/r/topic/61gi41uibbWvXjO56bj19q
Leave group <email obscured>?Subject=Unsubscribe
Hello there
Is anyone having success at profiling lysine acetylated peptides using an
immunoprecipitation enrichment strategy? Would anyone be willing to share their
experience of which commercially available antibodies and IP approaches work
well (or indeed do not work well) in their hands? The literature is the
literature but direct comments on these types of approaches are always more
useful in my opinion :-)
Many thanks in advance for help