You may find information in the following reference to be enlightening.
[cid:4fae025c-987c-4255-a1d4-4d726537f488]
Beverly DaGue
Has anybody updated mMass (mMass.org<http://mMass.org>) so the new Mac OS
Catalina can run it?
MMV
Martha M. Vestling, PhD
Chemistry Department
University of Wisconsin-Madison
<email obscured><email obscured>>
Hi Kym,
Check a water and reagent blank. I have found measurable levels of G and E in
distilled water stored in carboys.
All the best,
-laurey
Weather Inconsequential: Cold with snow flurries here. We may have a lot of
candy left over tomorrow night!
Laurey Steinke, Ph.D
Associate Professor
Department of Biochemistry and Molecular Biology
Co-Director, BMB Masters Program
Director, Protein Structure Core Facility
985819 Nebraska Medical Center
Omaha, NE 68198-5819
T: (402) 559-5176
F: (402) 559-6646
http://www.unmc.edu/biochemistry/education/master/index.html
http://www.unmc.edu/pscf/
๏ปฟOn 10/30/19, 4:58 PM, "ABRF Discussion Forum on behalf of Hampton, Brian"
<email obscured> on behalf of <email obscured>> wrote:
Non-UNMC email
Hi Kym,
When we did PicoTag amino acid analysis, we used Corning 5 x 50 mm Pyrex
culture tubes cat. number 9820. These glass tubes were pyrolized at ~~ 300
deg. C ( maybe higher, can't find the temp we used) to carbonize organics.
Samples were dried in these tubes and processed for AAA. By doing the
pyrolysis we at least knew any source of contamination was not coming from
those tubes.
Good luck,
Brian
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Kym Faull
<email obscured>>
Sent: Wednesday, October 30, 2019 5:39 PM
To: <email obscured> <email obscured>>
Cc: Alex Yoon <email obscured>>
Subject: [ABRF Discussion Forum] Low level amino acid analyses
We are attempting low level amino acid quantitation by LC/MS/MS-MRM. Our
work is plagued by an unacceptably high background for certain amino acids (G,
E, etc). We are looking for advice as to the most likely sources of
contamination of amino acids. Samples in micro centrifuge tubes (including
blanks) are dried in a speed vac, redissolved in an aqueous buffer to which is
added a reagent. Following reaction the samples are transferred to HPLC
injector vials and injected in to the LC/MS. All samples including the blanks
have a variable and at times big signals for some amino acids. We seek advice
as to the likley source(s) of contamination. Any and all advice will be
appreciated
Kym
Kym Francis Faull, Ph.D.
Director, Pasarow Mass Spectrometry Laboratory
Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA
Professor Emeritus, Department of Psychiatry and Biobehavioral Sciences
David Geffen School of Medicine at UCLA
Telephone: (310) 206 7881 (office)
Telephone: (310) 206 7886 (laboratory)
Facsimile: (310) 206 2161
Electronic mail: <email obscured> <email obscured>>
Hi Kym,
When we did PicoTag amino acid analysis, we used Corning 5 x 50 mm Pyrex
culture tubes cat. number 9820. These glass tubes were pyrolized at ~~ 300
deg. C ( maybe higher, can't find the temp we used) to carbonize organics.
Samples were dried in these tubes and processed for AAA. By doing the
pyrolysis we at least knew any source of contamination was not coming from
those tubes.
Good luck,
Rest of post
Brian
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Kym Faull
<email obscured>>
Sent: Wednesday, October 30, 2019 5:39 PM
To: <email obscured> <email obscured>>
Cc: Alex Yoon <email obscured>>
Subject: [ABRF Discussion Forum] Low level amino acid analyses
We are attempting low level amino acid quantitation by LC/MS/MS-MRM. Our work
is plagued by an unacceptably high background for certain amino acids (G, E,
etc). We are looking for advice as to the most likely sources of contamination
of amino acids. Samples in micro centrifuge tubes (including blanks) are
dried in a speed vac, redissolved in an aqueous buffer to which is added a
reagent. Following reaction the samples are transferred to HPLC injector vials
and injected in to the LC/MS. All samples including the blanks have a variable
and at times big signals for some amino acids. We seek advice as to the likley
source(s) of contamination. Any and all advice will be appreciated
Kym
Kym Francis Faull, Ph.D.
Director, Pasarow Mass Spectrometry Laboratory
Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA
Professor Emeritus, Department of Psychiatry and Biobehavioral Sciences
David Geffen School of Medicine at UCLA
Telephone: (310) 206 7881 (office)
Telephone: (310) 206 7886 (laboratory)
Facsimile: (310) 206 2161
Electronic mail: <email obscured> <email obscured>>
โโ
View topic http://list.abrf.org/r/topic/4WeIDWie35kScafQTpnh8F
Leave group <email obscured>?Subject=Unsubscribe
We are attempting low level amino acid quantitation by LC/MS/MS-MRM. Our work
is plagued by an unacceptably high background for certain amino acids (G, E,
etc). We are looking for advice as to the most likely sources of contamination
of amino acids. Samples in micro centrifuge tubes (including blanks) are
dried in a speed vac, redissolved in an aqueous buffer to which is added a
reagent. Following reaction the samples are transferred to HPLC injector vials
and injected in to the LC/MS. All samples including the blanks have a variable
and at times big signals for some amino acids. We seek advice as to the likley
source(s) of contamination. Any and all advice will be appreciated
Kym
Kym Francis Faull, Ph.D.
Director, Pasarow Mass Spectrometry Laboratory
Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA
Professor Emeritus, Department of Psychiatry and Biobehavioral Sciences
David Geffen School of Medicine at UCLA
Telephone: (310) 206 7881 (office)
Telephone: (310) 206 7886 (laboratory)
Facsimile: (310) 206 2161
Electronic mail: <email obscured> <email obscured>>
Personally I never had issues with A columns so I keep using them (Do I
need to sign a waiver :)) and luckily I always have 2-3 spares. I was told
itโs going to take 6-8 weeks. Meanwhile Thermo rep said they can sell me
the easy nano emitter at lowest possible price so that I can use other
columns.
Rest of post
Bin
On Wed, Oct 23, 2019 at 1:28 PM Christine Vogel <email obscured>> wrote:
> Hello,
>
> How is everyone dealing with Thermo's recall of the Easy LC columns (safety
> issue)? Alternatives?
>
> Thank you,
>
> Christine
>
> --
>
> Christine Vogel, PhD
> New York University - Department of Biology
> Center for Genomics & Systems Biology
> 12 Waverly Pl - Office #403
> New York, NY 10003
> phone: +1-212-998-3976
>
> https://vogellab.bio.nyu.edu/ --- @CVogelNYC
>
> โIf your dreams don't scare you, they are not big enoughโ Ellen Johnson
> Sirleaf
>
> โโ
> View topic http://list.abrf.org/r/topic/3iMEbvK2IobfgzvEr5RwPM
> Leave group <email obscured>?Subject=Unsubscribe
>
>
Hi Andy,
That is truly wonderful! Thanks a bunch for your invaluable input and help.
Much appreciated.
Rest of post
Sincerely,
Brian
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of
<email obscured> <email obscured>>
Sent: Friday, October 25, 2019 2:38 PM
To: <email obscured> <email obscured>>
Cc: 'Barry Boyes' <email obscured>>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Follow-up: Barry Boyes is now preparing a white paper several pages long on
the subject, complete with links to helpful posters and papers on the subject.
I'll post it on this forum once it's ready.
Andy Alpert
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of
<email obscured>
Sent: Friday, October 25, 2019 1:23 PM
To: <email obscured>
Cc: 'Barry Boyes' <email obscured>>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hi, Brian,
Judging from your response and Kym Faull's (in a separate e-mail, he's just
declared that he intends to try DFA), it might be appropriate to have a
mini-symposium on the uses of DFA. Considering how pervasive the use of TFA is
in chromatography and MS, that wouldn't be out of line on this forum. At this
point, perhaps it would be appropriate to invoke Barry Boyes himself. I'm
copying this to him and will see if I can get him online.
Andy Alper
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Brian Hampton
Sent: Friday, October 25, 2019 1:14 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hi Andy,
Thanks for the pointers to the Boyes work. WRT posters at ASMS, the sheer
volume overwhelms me (needle and haystack come to mind, although the haystack
doesn't come with a search function), not to mention that travel expenses limit
my attendance at most meetings outside the Washington/Baltimore region.
I recall that it was in 1984 when I performed my first HPLC chromatographic
separation. In all the years since until today I have regrettably never heard
of DFA. Makes me shudder at what else I've been missing.
Regards,
Brian
โโ
View topic http://list.abrf.org/r/topic/1rVUd3tumUCLuoyH0UVNzL
Leave group <email obscured>?Subject=Unsubscribe
โโ
View topic http://list.abrf.org/r/topic/3gXpSCf56VhDTe00w0GYAF
Leave group <email obscured>?Subject=Unsubscribe
โโ
View topic http://list.abrf.org/r/topic/MKpO0yoFjJlB8qyvZl3pg
Leave group <email obscured>?Subject=Unsubscribe
Follow-up: Barry Boyes is now preparing a white paper several pages long on
the subject, complete with links to helpful posters and papers on the subject.
I'll post it on this forum once it's ready.
Andy Alpert
Rest of post
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of
<email obscured>
Sent: Friday, October 25, 2019 1:23 PM
To: <email obscured>
Cc: 'Barry Boyes' <email obscured>>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hi, Brian,
Judging from your response and Kym Faull's (in a separate e-mail, he's just
declared that he intends to try DFA), it might be appropriate to have a
mini-symposium on the uses of DFA. Considering how pervasive the use of TFA is
in chromatography and MS, that wouldn't be out of line on this forum. At this
point, perhaps it would be appropriate to invoke Barry Boyes himself. I'm
copying this to him and will see if I can get him online.
Andy Alper
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Brian Hampton
Sent: Friday, October 25, 2019 1:14 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hi Andy,
Thanks for the pointers to the Boyes work. WRT posters at ASMS, the sheer
volume overwhelms me (needle and haystack come to mind, although the haystack
doesn't come with a search function), not to mention that travel expenses limit
my attendance at most meetings outside the Washington/Baltimore region.
I recall that it was in 1984 when I performed my first HPLC chromatographic
separation. In all the years since until today I have regrettably never heard
of DFA. Makes me shudder at what else I've been missing.
Regards,
Brian
โโ
View topic http://list.abrf.org/r/topic/1rVUd3tumUCLuoyH0UVNzL
Leave group <email obscured>?Subject=Unsubscribe
โโ
View topic http://list.abrf.org/r/topic/3gXpSCf56VhDTe00w0GYAF
Leave group <email obscured>?Subject=Unsubscribe
Hi, Brian,
Judging from your response and Kym Faull's (in a separate e-mail, he's just
declared that he intends to try DFA), it might be appropriate to have a
mini-symposium on the uses of DFA. Considering how pervasive the use of TFA is
in chromatography and MS, that wouldn't be out of line on this forum. At this
point, perhaps it would be appropriate to invoke Barry Boyes himself. I'm
copying this to him and will see if I can get him online.
Andy Alper
Rest of post
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Brian Hampton
Sent: Friday, October 25, 2019 1:14 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hi Andy,
Thanks for the pointers to the Boyes work. WRT posters at ASMS, the sheer
volume overwhelms me (needle and haystack come to mind, although the haystack
doesn't come with a search function), not to mention that travel expenses limit
my attendance at most meetings outside the Washington/Baltimore region.
I recall that it was in 1984 when I performed my first HPLC chromatographic
separation. In all the years since until today I have regrettably never heard
of DFA. Makes me shudder at what else I've been missing.
Regards,
Brian
โโ
View topic http://list.abrf.org/r/topic/1rVUd3tumUCLuoyH0UVNzL
Leave group <email obscured>?Subject=Unsubscribe
Hi Andy,
Thanks for the pointers to the Boyes work. WRT posters at ASMS, the sheer
volume overwhelms me (needle and haystack come to mind, although the haystack
doesn't come with a search function), not to mention that travel expenses limit
my attendance at most meetings outside the Washington/Baltimore region.
I recall that it was in 1984 when I performed my first HPLC chromatographic
separation. In all the years since until today I have regrettably never heard
of DFA. Makes me shudder at what else I've been missing.
Hi, Kym,
Well, if cognoscenti such as Brian Hampton donโt pick up on advances in method
from posters at ASMS and Barry Boyesโ papers in Journal of Chromatography A,
then it might be too much to hope that everyone else in the proteomics and mass
spec fields would have noticed them too. Iโll say this for Waters: While they
may not introduce new methods like this, they have the network to get them
adopted widely. It pays to advertise, in science as in all else.
All the best,
Andy Alpert
PolyLC Inc.
From: Kym Faull <email obscured>>
Sent: Friday, October 25, 2019 12:16 PM
To: <email obscured>
Cc: <email obscured>; Julian Whitelegge <email obscured>>; Whitaker Cohn
<email obscured>>; Joe Capri <email obscured>>; austin quach
<email obscured>>; Alex Yoon <email obscured>>; Alan Wagner
<email obscured>>; Lucy Gao <email obscured>>; annie Tagvoryan
<email obscured>>; Jaime Bulkacz <email obscured>>; Erdim Sertoglu
<email obscured>>; Adrian Gomez <email obscured>>; Brian Kawahara
<email obscured>>; Sarah Bui <email obscured>>
Subject: Re: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Andy: If all this isn true why hasnโt DFA caught on and be used more widely?
Kym
Kym Francis Faull, Ph.D.
Director, Pasarow Mass Spectrometry Laboratory
Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA
Professor Emeritus, Department of Psychiatry and Biobehavioral Sciences
David Geffen School of Medicine at UCLA
Telephone: (310) 206 7881 (office)
Telephone: (310) 206 7886 (laboratory)
Facsimile: (310) 206 2161
Electronic mail: <email obscured> <email obscured>>
Rest of post
On Oct 25, 2019, at 8:46 AM, <email obscured> <email obscured>> >
<email obscured> <email obscured>> > wrote:
Barry Boyes and his colleagues at Advanced Materials Technology were writing
about the virtues of DFA a couple of years before Waters became interested in
it. Perform a search on Google Scholar using "difluoroacetic" as a search term
and with "BE Boyes" as an author, from 2015 to the present. They've presented
several posters at ASMS conferences that describe the properties of DFA in
chromatography and MS, in great detail. Briefly, DFA is intermediate between
TFA and formic acid in its strength as an acid and the degree to which it
suppresses ionization in MS. It's transparent at low wavelengths. It's also
not unduly toxic (unlike fluoroacetic acid, which is toxic enough for use as a
chemical warfare agent).
Andy Alpert
PolyLC Inc.
-----Original Message-----
From: ABRF Discussion Forum <email obscured> <email obscured>> >
On Behalf Of Brian Hampton
Sent: Friday, October 25, 2019 11:31 AM
To: <email obscured> <email obscured>>
Subject: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hello all,
In a roundabout way I came across difluoroacetic acid (DFA) for improving
chromatographic separations - as compared to formic acid and trifluoroacetic
acid (TFA). After some digging I came across a poster from Waters directly
comparing peptide separations between FA, DFA and TFA. It looks like DFA
doesn't have the ion suppression effect of TFA, and like TFA, DFA improves peak
separation and peak capacity (sharper peaks) compared to FA. See:
https://www.waters.com/webassets/cms/library/docs/2019hplc_nguyen_dfapeptides.pdf
I'm wondering whether anyone else has tried DFA and also if it does work as
advertised I'm surprised it hasn't replaced the use of FA by now. I've never
seen DFA mentioned in proteomics papers.
Regards,
Brian
โโ
View topic http://list.abrf.org/r/topic/2qPCWiE3JrYGeMaLroF665
Leave group <email obscured>?Subject=Unsubscribe
โโ
View topic http://list.abrf.org/r/topic/1WBcttUm538S0VAluZAuUr
Leave group <email obscured>?Subject=Unsubscribe
Andy: If all this isn true why hasnโt DFA caught on and be used more widely?
Kym
Kym Francis Faull, Ph.D.
Director, Pasarow Mass Spectrometry Laboratory
Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA
Professor Emeritus, Department of Psychiatry and Biobehavioral Sciences
David Geffen School of Medicine at UCLA
Telephone: (310) 206 7881 (office)
Telephone: (310) 206 7886 (laboratory)
Facsimile: (310) 206 2161
Electronic mail: <email obscured> <email obscured>>
Rest of post
> On Oct 25, 2019, at 8:46 AM, <email obscured>> <email obscured>> wrote:
>
> Barry Boyes and his colleagues at Advanced Materials Technology were writing
about the virtues of DFA a couple of years before Waters became interested in
it. Perform a search on Google Scholar using "difluoroacetic" as a search term
and with "BE Boyes" as an author, from 2015 to the present. They've presented
several posters at ASMS conferences that describe the properties of DFA in
chromatography and MS, in great detail. Briefly, DFA is intermediate between
TFA and formic acid in its strength as an acid and the degree to which it
suppresses ionization in MS. It's transparent at low wavelengths. It's also
not unduly toxic (unlike fluoroacetic acid, which is toxic enough for use as a
chemical warfare agent).
>
> Andy Alpert
> PolyLC Inc.
>
> -----Original Message-----
> From: ABRF Discussion Forum <email obscured>> On Behalf Of Brian Hampton
> Sent: Friday, October 25, 2019 11:31 AM
> To: <email obscured>
> Subject: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
>
> Hello all,
>
> In a roundabout way I came across difluoroacetic acid (DFA) for improving
chromatographic separations - as compared to formic acid and trifluoroacetic
acid (TFA). After some digging I came across a poster from Waters directly
comparing peptide separations between FA, DFA and TFA. It looks like DFA
doesn't have the ion suppression effect of TFA, and like TFA, DFA improves peak
separation and peak capacity (sharper peaks) compared to FA. See:
https://www.waters.com/webassets/cms/library/docs/2019hplc_nguyen_dfapeptides.pdf
>
> I'm wondering whether anyone else has tried DFA and also if it does work as
advertised I'm surprised it hasn't replaced the use of FA by now. I've never
seen DFA mentioned in proteomics papers.
>
> Regards,
>
> Brian
>
>
> โโ
> View topic http://list.abrf.org/r/topic/2qPCWiE3JrYGeMaLroF665
> Leave group <email obscured>?Subject=Unsubscribe
>
>
>
> โโ
> View topic http://list.abrf.org/r/topic/1WBcttUm538S0VAluZAuUr
> Leave group <email obscured>?Subject=Unsubscribe
>
Hello all,
Do any of you still perform Amino acid analysis using post-column
derivatization with Ninhydrin? What about Edman degradation?
The Protein Structure Core Facility at UNMC will be closing at the end of this
fiscal year, after 30 years of operation, and I am looking for good places to
recommend to my customers.
It has been a fantastic run. For me the best part was the incredible people I
met in the ABRF, followed in close second by the customers who brought me their
interesting problems to solve. I will miss all of you and all of them. I am
not retiring yet. Still teaching and running a research lab, but I will miss
my involvement with core facilities and the wonderful people who run them.
If you are coming through Nebraska, let me know!
All the best,
-laurey
Weather Inconsequential: It is a crisp clear early fall day. Leaves are
turning and I have pulled out my sweaters. It will be a good afternoon to walk
my dog if I can get home early enough-the light is fading early these days.
Laurey Steinke, Ph.D
Associate Professor
Department of Biochemistry and Molecular Biology
Co-Director, BMB Masters Program
Director, Protein Structure Core Facility
985819 Nebraska Medical Center
Omaha, NE 68198-5819
T: (402) 559-5176
F: (402) 559-6646
http://www.unmc.edu/biochemistry/education/master/index.html
http://www.unmc.edu/pscf/
The information in this e-mail may be privileged and confidential, intended
only for the use of the addressee(s) above. Any unauthorized use or disclosure
of this information is prohibited. If you have received this e-mail by mistake,
please delete it and immediately contact the sender.
Barry Boyes and his colleagues at Advanced Materials Technology were writing
about the virtues of DFA a couple of years before Waters became interested in
it. Perform a search on Google Scholar using "difluoroacetic" as a search term
and with "BE Boyes" as an author, from 2015 to the present. They've presented
several posters at ASMS conferences that describe the properties of DFA in
chromatography and MS, in great detail. Briefly, DFA is intermediate between
TFA and formic acid in its strength as an acid and the degree to which it
suppresses ionization in MS. It's transparent at low wavelengths. It's also
not unduly toxic (unlike fluoroacetic acid, which is toxic enough for use as a
chemical warfare agent).
Andy Alpert
PolyLC Inc.
Rest of post
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Brian Hampton
Sent: Friday, October 25, 2019 11:31 AM
To: <email obscured>
Subject: [ABRF Discussion Forum] DFA as an ion pair agent for LC/MS
Hello all,
In a roundabout way I came across difluoroacetic acid (DFA) for improving
chromatographic separations - as compared to formic acid and trifluoroacetic
acid (TFA). After some digging I came across a poster from Waters directly
comparing peptide separations between FA, DFA and TFA. It looks like DFA
doesn't have the ion suppression effect of TFA, and like TFA, DFA improves peak
separation and peak capacity (sharper peaks) compared to FA. See:
https://www.waters.com/webassets/cms/library/docs/2019hplc_nguyen_dfapeptides.pdf
I'm wondering whether anyone else has tried DFA and also if it does work as
advertised I'm surprised it hasn't replaced the use of FA by now. I've never
seen DFA mentioned in proteomics papers.
Regards,
Brian
โโ
View topic http://list.abrf.org/r/topic/2qPCWiE3JrYGeMaLroF665
Leave group <email obscured>?Subject=Unsubscribe
Hello Chris,
Some info here. See: https://dx.doi.org/10.1021/acs.jproteome.8b00831
Baud et al., โAn Optimized Method for the Proteomic Analysis of Low Volumes of
Cell Culture Media and the Secretome: The Application and the Demonstration of
Altered Protein Expression in iPSC-Derived Neuronal Cell Lines from Parkinsonโs
Disease Patients.โ
For our own purposes, we simply rinse cells 3x with cold PBS and move on with a
proteomics workflow. The above paper has some interesting observations that
indicate culture medium proteins continue to be released from the cells over
time.
Hello all,
In a roundabout way I came across difluoroacetic acid (DFA) for improving
chromatographic separations - as compared to formic acid and trifluoroacetic
acid (TFA). After some digging I came across a poster from Waters directly
comparing peptide separations between FA, DFA and TFA. It looks like DFA
doesn't have the ion suppression effect of TFA, and like TFA, DFA improves peak
separation and peak capacity (sharper peaks) compared to FA. See:
https://www.waters.com/webassets/cms/library/docs/2019hplc_nguyen_dfapeptides.pdf
I'm wondering whether anyone else has tried DFA and also if it does work as
advertised I'm surprised it hasn't replaced the use of FA by now. I've never
seen DFA mentioned in proteomics papers.
I was wondering what procedure you use for extracting proteins from cells in
culture for proteomics analyses.
Specifically, I am curious how one avoids proteins from the cell culture media
that might be sticking to the cells and interfere with mass spectrometry
analysis.
After removing cell culture media how do you wash cells to remove media
proteins that are sticking to the cells/binding to cell surface proteins and
lipids?
Thanks much!
I am blissfully unaware of it! I think it it okay for you to put a link to
something like that.
D. Eric Anderson
Mass Spectrometry Facility, NIDDK
NIH
Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
(301)-496-7546
Rest of post
________________________________________
From: Christine Vogel <email obscured>>
Sent: 23 October 2019 13:27
To: <email obscured>
Subject: [ABRF Discussion Forum] Thermo Easy LC column issue
Hello,
How is everyone dealing with Thermo's recall of the Easy LC columns (safety
issue)? Alternatives?
Thank you,
Christine
--
Christine Vogel, PhD
New York University - Department of Biology
Center for Genomics & Systems Biology
12 Waverly Pl - Office #403
New York, NY 10003
phone: +1-212-998-3976
https://vogellab.bio.nyu.edu/ --- @CVogelNYC
โIf your dreams don't scare you, they are not big enoughโ Ellen Johnson
Sirleaf
โโ
View topic http://list.abrf.org/r/topic/3iMEbvK2IobfgzvEr5RwPM
Leave group <email obscured>?Subject=Unsubscribe
Hello,
How is everyone dealing with Thermo's recall of the Easy LC columns (safety
issue)? Alternatives?
Thank you,
Rest of post
Christine
--
Christine Vogel, PhD
New York University - Department of Biology
Center for Genomics & Systems Biology
12 Waverly Pl - Office #403
New York, NY 10003
phone: +1-212-998-3976
https://vogellab.bio.nyu.edu/ --- @CVogelNYC
โIf your dreams don't scare you, they are not big enoughโ Ellen Johnson
Sirleaf
Hello Yishai,
In adding on to what Will mentioned is another paper by Shuford "Absolute
Protein Quantification by Mass Spectrometry: Not as Simple as Advertised" (DOI:
10.1021/acs.analchem.7b00858) where they discuss the reasons and show data on
how variable absolute quantification is - depending on what you use for your
standards. Their conclusion is that SIL proteins as internal standards are
preferable to peptide standards.
Choosing which peptides to use as surrogates for protein concentration is not
easy. Digestibility of peptides will result in differences in the absolute
amount of each peptide being quantified, as will other physical properties such
as whether it sticks to surfaces more so than another peptide or is more/less
susceptible to artifactual PTM that splits the signal for that peptide among
multiple forms.
AP3 is a tool reported to help select target peptides as it incorporates
digestibility and detectability. See: "AP3: an advanced proteotypic peptide
predictor for targeted proteomics by incorporating peptide digestibility". DOI:
10.1021/acs.analchem.9b02520.
You might also use a DDA experiment on the samples and search them with
MetaMorpheus (with G-PTM-D) to see whether any of your selected peptides
contain modifications that you have not accounted for. I would not use urea at
all in the work flow (carbamylation), but instead use SDC or dodecylmaltoside
in necessary. Halogenated alkylating reagents can induce quite a bit of side
chain modifications as well.
Regards,
Brian
Brian Hampton
Protein Analysis Lab
Center for Vascular and Inflammatory Diseases
University of Maryland School of Medicine
BioPark One Rm 301
800 West Baltimore Street
Baltimore, MD. 21201
Rest of post
(410)706-8207
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of J. Will Thompson,
Ph.D. <email obscured>>
Sent: Wednesday, October 23, 2019 2:53 AM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] Absolute quan using targeted proteomics
Okay, Yishai, Iโll bite.
My opinion is that this is a very common occurrence that nobody likes to talk
about. The heavy peptide spike is very good at quantifying the amount of native
peptide in your sample at the time of injection, and actually pretty poor at
giving you the amount of the native protein originally in your sample. The
reasons are many, including enzymatic inefficiency/incompleteness, peptide
degradation, over digestion, protein modification, amino acid substitution
(maybe not in your case), among others.
Shuford and Muddiman, MCP 2012 (โPeptide production and decay rates...) gives a
lot of useful information.
Any time we want to quantify a protein by MS, we now use SIL but produce the
curve to quantify against either a reference standard (such as purified
protein) or a single point calibration against a reference pool (like a pooled
plasma). An example of protein quant using this approach (this is just one of
our collaborations, there are certainly other good ones out there) is Schaller
TH et al J Protein Res, 2019.
Would love to hear if others are out there with similar or different
experience!
Cheers
W
Sent from my iPhone
> On Oct 23, 2019, at 7:02 AM, Yishai Levin <email obscured>>
wrote:
>
> ๏ปฟGreetings,
> We ran into some trouble while developing a PRM assay for absolute
quantification of 3 (abundant) proteins in e. coli. We Have 3 synthetic
peptides per protein, with heavy stable isotopes (sourced from JPT with their
QTag).
> We spike the peptides before digestion, into the lysis buffer (8M urea,
diluted prior to tryptic digestion).
> We made a 10 point calibration curve for each peptide and included
replicates. We got good linearity for all peptides and good precision.
> The problem is that each peptide produces a different absolute concentration,
the differences being as large as 5 fold (!) for the same protein.
> Has anyone encountered this problem before? Any thoughts on how to
troubleshoot it?
>
> Thanks,
>
> Yishai
>
> Yishai Levin, PhD
> Head of the de Botton Institute for Protein Profiling
> The Nancy and Stephen Grand
> Israel National Center for Personalised Medicine
> Weizmann Institute of Science, Rehovot 76100, Israel
> Tel. 972-8-9344315
> Our
website<https://urldefense.proofpoint.com/v2/url?u=http-3A__g-2Dincpm.weizmann.ac.il_protein-2Dprofiling_de-2Dbotton-2Dinstitute-2Dprotein-2Dprofiling&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=1yQnHeg5Q5znzQN9_6TbOdt3nCSpUcvJdBMFoCLg5uA&s=KtEoggSQVXvkqFVzDGtDqHhpnxg9wcR0IJOJJ17T5DM&e=
>
>
>
>
>
>
> โโ
> View topic
https://urldefense.proofpoint.com/v2/url?u=http-3A__list.abrf.org_r_topic_7yj34PMN0IjqnZqwe8qHlG&d=DwIFaQ&c=imBPVzF25OnBgGmVOlcsiEgHoG1i6YHLR0Sj_gZ4adc&r=UBQRX1RXWAgFeW3VEViBQCYaC3oYasD8J7zLepPz1Cw&m=1yQnHeg5Q5znzQN9_6TbOdt3nCSpUcvJdBMFoCLg5uA&s=nKrS4jPCWqn05FnAZhT3QCRgFCqcHoubxm9l5PwexMA&e=
> Leave group <email obscured>?Subject=Unsubscribe
>
โโ
View topic http://list.abrf.org/r/topic/5KUYSUTHctDgSoL4WHbq0j
Leave group <email obscured>?Subject=Unsubscribe