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The Committee for Core Rigor and Reproducibility (CCoRRe):
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Okay this is probably not going to work but it is easy to try.
On **the same computer** as where you collected the data copy the file (don't
move it - make a copy) from its original location to another. If not from one
drive to another then at least to a separate folder or attach an external drive
and copy it to that drive. This used to be the solution back aways for this
sort of issue but I do seem to recall some limits on file size as mentioned
previously.
Eric
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: Yishai Levin <email obscured>>
Sent: 08 February 2021 02:32
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Problems viewing large data files in Qual
Browzer
Hi Kym,
Try converting the data to mzML using msconvert (ProteoWizard). Maybe you’ll be
able to open them after the conversion.
Yishai
> On 3 Feb 2021, at 20:44, Kym Faull <email obscured>> wrote:
>
> Colleagues: On an old Orbitrap instrument I collected some large LC/MS data
files in the ion trap negative and positive ion modes. The two modes (+ and -)
were done on the same samples with the same column and almost identical eluants
(0.1% formic for + and 0.01% formic for -). The negative data files are about
960,000 kB in size and they can be accessed using Qual Browzer on the
instrument with which they were collected and on another instrument after
copying the files over. The + mode data files are about 2,600,000 kB in size
and so far I have been unable to open them in Qual Browzer on either the
instrument with which they were collected or on another instrument after
copying the files over. Is the problem with the + mode date files their size?
Is there some setting that needs to be adjusted before they can be viewed? Can
anyone provide advice about this problem? 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 on Recall, 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>>
>
>
>
>
>
>
>
>
>
> ――
> View topic http://list.abrf.org/r/topic/2jGKZJTo5F6KDjKCqJFu9
> Leave group <email obscured>?Subject=Unsubscribe
>
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Leave group <email obscured>?Subject=Unsubscribe
Hi Kym,
Try converting the data to mzML using msconvert (ProteoWizard). Maybe you’ll be
able to open them after the conversion.
Yishai
> On 3 Feb 2021, at 20:44, Kym Faull <email obscured>> wrote:
>
> Colleagues: On an old Orbitrap instrument I collected some large LC/MS data
files in the ion trap negative and positive ion modes. The two modes (+ and -)
were done on the same samples with the same column and almost identical eluants
(0.1% formic for + and 0.01% formic for -). The negative data files are about
960,000 kB in size and they can be accessed using Qual Browzer on the
instrument with which they were collected and on another instrument after
copying the files over. The + mode data files are about 2,600,000 kB in size
and so far I have been unable to open them in Qual Browzer on either the
instrument with which they were collected or on another instrument after
copying the files over. Is the problem with the + mode date files their size?
Is there some setting that needs to be adjusted before they can be viewed? Can
anyone provide advice about this problem? 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 on Recall, Department of Psychiatry and Biobehavioral
Sciences
Rest of post
> 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>>
>
>
>
>
>
>
>
>
>
> ――
> View topic http://list.abrf.org/r/topic/2jGKZJTo5F6KDjKCqJFu9
> Leave group <email obscured>?Subject=Unsubscribe
>
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Have you tried opening the file in Freestyle? This is the replacement for
xcalibur qual browser. Maybe that can cope with the larger file size? Both
Xcalibur and included freestyle can be downloaded from Thermo’s flexnet site.
thermo.flexnetoperations.com<http://thermo.flexnetoperations.com> where you can
register for a free account.
Alternatively if end of file corruption is the issue then the freeware
MSFilereader should allow you to extract just the earlier portion of the file
(you can select start and end scans).
Rest of post
Steve
------------------------------------------------
Stephen J. Eyles, D.Phil. (he/him/his)
Extension Professor, Biochemistry & Molecular Biology
Director, Mass Spectrometry Core, Institute for Applied Life Sciences
University of Massachusetts Amherst
240 Thatcher Rd
S503 Life Science Laboratories
Amherst, MA 01003-9364
Tel: (413) 577-1528
Cell: (413) 297-6709
E-mail: <email obscured><email obscured>>
http://www.umass.edu/ials/mass-spectrometry
------------------------------------------------
On Feb 3, 2021, at 4:29 PM, Lauri Peil
<email obscured><email obscured>>> wrote:
Hi all
To the best of my knowledge newer Xcalibur will not help you, these files are
corrupted and end of story. Older versions of Xcaliburs were capable of writing
larger than 2 GB files but they didn't close them correctly, therefore at the
end of acquisition you lost all the data. I mean, in principle the data are
still there, it's just that there are no means of accessing them...
Thermo may have come up with a solution lately, few years ago it was like that,
I myself lost a good few runs...
Lauri
On 3 February 2021 21:31:06 EET, "Hampton, Brian"
<email obscured><email obscured>>> wrote:
Hi Kym,
I think the problem you have is with a limitation of the
XCalibur/Qualbrowser software version that isn't capable of reading
files larger than 2Gb. You should be able to obtain a newer version of
XCalibur which will read these files.
The large file sizes may be due to being collected in profile mode
rather than centroid. If profile data isn't necessary, then you can
collect the data in centroid mode so that the file size will be much
smaller. In the case of shotgun proteomics runs on the Orbitrap
instruments, the MS1 scans are typically collected in the orbitrap in
profile mode and the MS2 scans in the ion trap in centroid mode and are
much smaller than collecting in all profile mode. Thermo Tech Support,
or your local Thermo Sales Rep. may be of help in getting you newer
XCalibur software - or another person at your institution may have a
newer version you can borrow and install on a separate PC for viewing
the files.
Best regards,
Brian
________________________________
From: ABRF Discussion Forum <email obscured><email obscured>>> on
behalf of Kym Faull
<email obscured><email obscured>>>
Sent: Wednesday, February 3, 2021 1:44 PM
To: <email obscured><email obscured>>
<email obscured><email obscured>>>
Subject: [ABRF Discussion Forum] Problems viewing large data files in
Qual Browzer
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.
Colleagues: On an old Orbitrap instrument I collected some large LC/MS
data files in the ion trap negative and positive ion modes. The two
modes (+ and -) were done on the same samples with the same column and
almost identical eluants (0.1% formic for + and 0.01% formic for -).
The negative data files are about 960,000 kB in size and they can be
accessed using Qual Browzer on the instrument with which they were
collected and on another instrument after copying the files over. The
+ mode data files are about 2,600,000 kB in size and so far I have been
unable to open them in Qual Browzer on either the instrument with which
they were collected or on another instrument after copying the files
over. Is the problem with the + mode date files their size? Is there
some setting that needs to be adjusted before they can be viewed? Can
anyone provide advice about this problem? 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 on Recall, 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>>
<email obscured>>
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Hi all
To the best of my knowledge newer Xcalibur will not help you, these files are
corrupted and end of story. Older versions of Xcaliburs were capable of writing
larger than 2 GB files but they didn't close them correctly, therefore at the
end of acquisition you lost all the data. I mean, in principle the data are
still there, it's just that there are no means of accessing them...
Thermo may have come up with a solution lately, few years ago it was like that,
I myself lost a good few runs...
Lauri
On 3 February 2021 21:31:06 EET, "Hampton, Brian" <email obscured>>
wrote:
Rest of post
>Hi Kym,
>
>I think the problem you have is with a limitation of the
>XCalibur/Qualbrowser software version that isn't capable of reading
>files larger than 2Gb. You should be able to obtain a newer version of
>XCalibur which will read these files.
>
>The large file sizes may be due to being collected in profile mode
>rather than centroid. If profile data isn't necessary, then you can
>collect the data in centroid mode so that the file size will be much
>smaller. In the case of shotgun proteomics runs on the Orbitrap
>instruments, the MS1 scans are typically collected in the orbitrap in
>profile mode and the MS2 scans in the ion trap in centroid mode and are
>much smaller than collecting in all profile mode. Thermo Tech Support,
>or your local Thermo Sales Rep. may be of help in getting you newer
>XCalibur software - or another person at your institution may have a
>newer version you can borrow and install on a separate PC for viewing
>the files.
>
>Best regards,
>Brian
>
>________________________________
>From: ABRF Discussion Forum <email obscured>> on behalf of Kym Faull
<email obscured>>
>Sent: Wednesday, February 3, 2021 1:44 PM
>To: <email obscured> <email obscured>>
>Subject: [ABRF Discussion Forum] Problems viewing large data files in
>Qual Browzer
>
>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.
>
>
>
>Colleagues: On an old Orbitrap instrument I collected some large LC/MS
>data files in the ion trap negative and positive ion modes. The two
>modes (+ and -) were done on the same samples with the same column and
>almost identical eluants (0.1% formic for + and 0.01% formic for -).
>The negative data files are about 960,000 kB in size and they can be
>accessed using Qual Browzer on the instrument with which they were
>collected and on another instrument after copying the files over. The
>+ mode data files are about 2,600,000 kB in size and so far I have been
>unable to open them in Qual Browzer on either the instrument with which
>they were collected or on another instrument after copying the files
>over. Is the problem with the + mode date files their size? Is there
>some setting that needs to be adjusted before they can be viewed? Can
>anyone provide advice about this problem? 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 on Recall, 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>>
>
>
>
>
>
>
>
>
>
>――
>View topic
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Sent from my Android device with K-9 Mail. Please excuse my brevity.
Hi Kym,
I think the problem you have is with a limitation of the XCalibur/Qualbrowser
software version that isn't capable of reading files larger than 2Gb. You
should be able to obtain a newer version of XCalibur which will read these
files.
The large file sizes may be due to being collected in profile mode rather than
centroid. If profile data isn't necessary, then you can collect the data in
centroid mode so that the file size will be much smaller. In the case of
shotgun proteomics runs on the Orbitrap instruments, the MS1 scans are
typically collected in the orbitrap in profile mode and the MS2 scans in the
ion trap in centroid mode and are much smaller than collecting in all profile
mode. Thermo Tech Support, or your local Thermo Sales Rep. may be of help in
getting you newer XCalibur software - or another person at your institution may
have a newer version you can borrow and install on a separate PC for viewing
the files.
Best regards,
Brian
Rest of post
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Kym Faull
<email obscured>>
Sent: Wednesday, February 3, 2021 1:44 PM
To: <email obscured> <email obscured>>
Subject: [ABRF Discussion Forum] Problems viewing large data files in Qual
Browzer
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.
Colleagues: On an old Orbitrap instrument I collected some large LC/MS data
files in the ion trap negative and positive ion modes. The two modes (+ and -)
were done on the same samples with the same column and almost identical eluants
(0.1% formic for + and 0.01% formic for -). The negative data files are about
960,000 kB in size and they can be accessed using Qual Browzer on the
instrument with which they were collected and on another instrument after
copying the files over. The + mode data files are about 2,600,000 kB in size
and so far I have been unable to open them in Qual Browzer on either the
instrument with which they were collected or on another instrument after
copying the files over. Is the problem with the + mode date files their size?
Is there some setting that needs to be adjusted before they can be viewed? Can
anyone provide advice about this problem? 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 on Recall, 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>>
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Colleagues: On an old Orbitrap instrument I collected some large LC/MS data
files in the ion trap negative and positive ion modes. The two modes (+ and -)
were done on the same samples with the same column and almost identical eluants
(0.1% formic for + and 0.01% formic for -). The negative data files are about
960,000 kB in size and they can be accessed using Qual Browzer on the
instrument with which they were collected and on another instrument after
copying the files over. The + mode data files are about 2,600,000 kB in size
and so far I have been unable to open them in Qual Browzer on either the
instrument with which they were collected or on another instrument after
copying the files over. Is the problem with the + mode date files their size?
Is there some setting that needs to be adjusted before they can be viewed? Can
anyone provide advice about this problem? 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 on Recall, 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>>
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For more details on the 2021 Poster Session, and to submit your abstract, visit
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ABRF Education Committee
The ABRF Education Committee is hosting these upcoming programs:
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February 9 & 11, 2021
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February 23 & 25, 2021
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For more information on these programs, and to register, visit the ABRF web
site.
https://abrf.org/upcoming-educational-opportunities
I missed your comment about the TIC higher in B. That points to erroneous
peptide assay results.
Brian Hampton
Proteomics Core 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 Hampton, Brian
<email obscured>>
Sent: Thursday, January 28, 2021 4:05 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] serum samples -- peptide quantification
vs. identification in the MS
Could there have been some procedural anomaly in A where an amine containing
component of the digest such as Tris was not completely removed? Regardless, I
would repeat the whole experiment and even use two different methods to
ascertain peptide recovery. That way you don’t move forward based on
potentially erroneous results from a peptide assay.
Brian Hampton
Proteomics Core Lab
Center for Vascular and Inflammatory Diseases
University of Maryland School of Medicine
BioPark One Rm 301
800 West Baltimore Street
Baltimore, MD. 21201
(410)706-8207
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Christine Vogel
<email obscured>>
Sent: Thursday, January 28, 2021 3:49 PM
To: <email obscured> <email obscured>>
Cc: Smruti D Pushalkar <email obscured>>
Subject: Re: [ABRF Discussion Forum] serum samples -- peptide quantification
vs. identification in the MS
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.
Great points (also from Steve Eyles on amine in the buffers...). We will
look into this...
Thank you! (I knew there is so much wisdom in the ABRF community...)
On Thu, Jan 28, 2021 at 3:05 PM Martin Middleditch <
<email obscured>> wrote:
> Hi Christine,
>
>
> Given that you are seeing similar numbers of proteins identified in both
> preps, but more peptides in B, is it possible that you are getting a
> difference in trypsin digestion efficiency such that B gives you a mixture
> of fully cleaved and missed cleaved peptides? Or alternatively, could
> protocol B be inducing more artefactual modifications (deamidation,
> oxidation, pyro-glu formations) than protocol A?
>
>
> Best regards,
>
> Martin
>
> Martin Middleditch
> Lead Technologist (Mass Spectrometry)
> School of Biological Sciences
> University of Auckland
> Room 301-422
> 23 Symonds Street
> Auckland
> (09) 923-7682 or 923-7540
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of Christine
> Vogel <email obscured>>
> Sent: Friday, January 29, 2021 8:56 AM
> To: <email obscured>; Smruti D Pushalkar
> Subject: Re: [ABRF Discussion Forum] serum samples -- peptide
> quantification vs. identification in the MS
>
> We used the Pierce Fluorometric Peptide Assay to estimate peptide
> concentration (labels N-terminal amines). That's another source for bias.
>
> TICs are higher for protocol B (the one with lower measured peptide
> concentration but higher identification in MS).
>
> I am tempted to go ahead with protocol B, but still puzzled about the
> higher apparent peptide concentration in protocol A.
>
> Best,
>
> Christine
>
>
>
> On Thu, Jan 28, 2021 at 2:50 PM Anderson, David Eric (NIH/NIDDK) [E] <
> <email obscured>> wrote:
>
> > Is your assumption about the difference in concentration of the material
> > born out in the TIC's. Does the overall look of the raw data look likes
> it
> > comes from the same amount of peptides?
> >
> > D. Eric Anderson, Ph.D
> > Mass Spectrometry Facility, NIDDK
> > NIH
> >
> >
> > Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
> > (301)-496-7546
> >
> >
> >
> > ________________________________________
> > From: Christine Vogel <email obscured>>
> > Sent: 28 January 2021 14:04
> > To: <email obscured>; Smruti D Pushalkar
> > Subject: [ABRF Discussion Forum] serum samples -- peptide quantification
> > vs. identification in the MS
> >
> > Dear Colleagues,
> >
> > I'm hoping to pick your brain on something that puzzles me: despite lower
> > overall peptide yield in one prep of serum samples vs. a different prep,
> > we observe higher identification of peptides in mass spec analysis.
> >
> > Long story: we are testing protocol A and B for tryptic digest etc of
> human
> > serum samples. In the same final elution volume (spin tip cleanup),
> > protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
> > for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
> > mode. We identify similar numbers of protein groups for protocol A and B,
> > BUT protocol B identifies more peptides and precursors.
> >
> > It seems like protocol A retrieves more peptides, but fewer *different*
> > peptides, i.e. it might just digest the highly abundant proteins such as
> > albumin more than protocol B?
> >
> > Both protocols A and B use a simple workflow of denaturation, alkylation,
> > tryptic digest and cleanup.
> >
> > 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
> > pronouns: she/her/hers
> >
> >
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> @CVogelNYC
> >
> > “If your dreams don't scare you, they are not big enough” Ellen Johnson
> > Sirleaf
> >
> > ――
> > View topic
> >
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> --
>
> 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
> pronouns: she/her/hers
>
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> @CVogelNYC
>
> “If your dreams don't scare you, they are not big enough” Ellen Johnson
> Sirleaf
>
> ――
> View topic
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--
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
pronouns: she/her/hers
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--- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
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Could there have been some procedural anomaly in A where an amine containing
component of the digest such as Tris was not completely removed? Regardless, I
would repeat the whole experiment and even use two different methods to
ascertain peptide recovery. That way you don’t move forward based on
potentially erroneous results from a peptide assay.
Brian Hampton
Proteomics Core 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 Christine Vogel
<email obscured>>
Sent: Thursday, January 28, 2021 3:49 PM
To: <email obscured> <email obscured>>
Cc: Smruti D Pushalkar <email obscured>>
Subject: Re: [ABRF Discussion Forum] serum samples -- peptide quantification
vs. identification in the MS
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.
Great points (also from Steve Eyles on amine in the buffers...). We will
look into this...
Thank you! (I knew there is so much wisdom in the ABRF community...)
On Thu, Jan 28, 2021 at 3:05 PM Martin Middleditch <
<email obscured>> wrote:
> Hi Christine,
>
>
> Given that you are seeing similar numbers of proteins identified in both
> preps, but more peptides in B, is it possible that you are getting a
> difference in trypsin digestion efficiency such that B gives you a mixture
> of fully cleaved and missed cleaved peptides? Or alternatively, could
> protocol B be inducing more artefactual modifications (deamidation,
> oxidation, pyro-glu formations) than protocol A?
>
>
> Best regards,
>
> Martin
>
> Martin Middleditch
> Lead Technologist (Mass Spectrometry)
> School of Biological Sciences
> University of Auckland
> Room 301-422
> 23 Symonds Street
> Auckland
> (09) 923-7682 or 923-7540
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of Christine
> Vogel <email obscured>>
> Sent: Friday, January 29, 2021 8:56 AM
> To: <email obscured>; Smruti D Pushalkar
> Subject: Re: [ABRF Discussion Forum] serum samples -- peptide
> quantification vs. identification in the MS
>
> We used the Pierce Fluorometric Peptide Assay to estimate peptide
> concentration (labels N-terminal amines). That's another source for bias.
>
> TICs are higher for protocol B (the one with lower measured peptide
> concentration but higher identification in MS).
>
> I am tempted to go ahead with protocol B, but still puzzled about the
> higher apparent peptide concentration in protocol A.
>
> Best,
>
> Christine
>
>
>
> On Thu, Jan 28, 2021 at 2:50 PM Anderson, David Eric (NIH/NIDDK) [E] <
> <email obscured>> wrote:
>
> > Is your assumption about the difference in concentration of the material
> > born out in the TIC's. Does the overall look of the raw data look likes
> it
> > comes from the same amount of peptides?
> >
> > D. Eric Anderson, Ph.D
> > Mass Spectrometry Facility, NIDDK
> > NIH
> >
> >
> > Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
> > (301)-496-7546
> >
> >
> >
> > ________________________________________
> > From: Christine Vogel <email obscured>>
> > Sent: 28 January 2021 14:04
> > To: <email obscured>; Smruti D Pushalkar
> > Subject: [ABRF Discussion Forum] serum samples -- peptide quantification
> > vs. identification in the MS
> >
> > Dear Colleagues,
> >
> > I'm hoping to pick your brain on something that puzzles me: despite lower
> > overall peptide yield in one prep of serum samples vs. a different prep,
> > we observe higher identification of peptides in mass spec analysis.
> >
> > Long story: we are testing protocol A and B for tryptic digest etc of
> human
> > serum samples. In the same final elution volume (spin tip cleanup),
> > protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
> > for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
> > mode. We identify similar numbers of protein groups for protocol A and B,
> > BUT protocol B identifies more peptides and precursors.
> >
> > It seems like protocol A retrieves more peptides, but fewer *different*
> > peptides, i.e. it might just digest the highly abundant proteins such as
> > albumin more than protocol B?
> >
> > Both protocols A and B use a simple workflow of denaturation, alkylation,
> > tryptic digest and cleanup.
> >
> > 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
> > pronouns: she/her/hers
> >
> >
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---
> @CVogelNYC
> >
> > “If your dreams don't scare you, they are not big enough” Ellen Johnson
> > Sirleaf
> >
> > ――
> > View topic
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> --
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> 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
> pronouns: she/her/hers
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---
> @CVogelNYC
>
> “If your dreams don't scare you, they are not big enough” Ellen Johnson
> Sirleaf
>
> ――
> View topic
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--
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
pronouns: she/her/hers
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--- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
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Great points (also from Steve Eyles on amine in the buffers...). We will
look into this...
Thank you! (I knew there is so much wisdom in the ABRF community...)
On Thu, Jan 28, 2021 at 3:05 PM Martin Middleditch <
Rest of post
<email obscured>> wrote:
> Hi Christine,
>
>
> Given that you are seeing similar numbers of proteins identified in both
> preps, but more peptides in B, is it possible that you are getting a
> difference in trypsin digestion efficiency such that B gives you a mixture
> of fully cleaved and missed cleaved peptides? Or alternatively, could
> protocol B be inducing more artefactual modifications (deamidation,
> oxidation, pyro-glu formations) than protocol A?
>
>
> Best regards,
>
> Martin
>
> Martin Middleditch
> Lead Technologist (Mass Spectrometry)
> School of Biological Sciences
> University of Auckland
> Room 301-422
> 23 Symonds Street
> Auckland
> (09) 923-7682 or 923-7540
> ________________________________
> From: ABRF Discussion Forum <email obscured>> on behalf of Christine
> Vogel <email obscured>>
> Sent: Friday, January 29, 2021 8:56 AM
> To: <email obscured>; Smruti D Pushalkar
> Subject: Re: [ABRF Discussion Forum] serum samples -- peptide
> quantification vs. identification in the MS
>
> We used the Pierce Fluorometric Peptide Assay to estimate peptide
> concentration (labels N-terminal amines). That's another source for bias.
>
> TICs are higher for protocol B (the one with lower measured peptide
> concentration but higher identification in MS).
>
> I am tempted to go ahead with protocol B, but still puzzled about the
> higher apparent peptide concentration in protocol A.
>
> Best,
>
> Christine
>
>
>
> On Thu, Jan 28, 2021 at 2:50 PM Anderson, David Eric (NIH/NIDDK) [E] <
> <email obscured>> wrote:
>
> > Is your assumption about the difference in concentration of the material
> > born out in the TIC's. Does the overall look of the raw data look likes
> it
> > comes from the same amount of peptides?
> >
> > D. Eric Anderson, Ph.D
> > Mass Spectrometry Facility, NIDDK
> > NIH
> >
> >
> > Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
> > (301)-496-7546
> >
> >
> >
> > ________________________________________
> > From: Christine Vogel <email obscured>>
> > Sent: 28 January 2021 14:04
> > To: <email obscured>; Smruti D Pushalkar
> > Subject: [ABRF Discussion Forum] serum samples -- peptide quantification
> > vs. identification in the MS
> >
> > Dear Colleagues,
> >
> > I'm hoping to pick your brain on something that puzzles me: despite lower
> > overall peptide yield in one prep of serum samples vs. a different prep,
> > we observe higher identification of peptides in mass spec analysis.
> >
> > Long story: we are testing protocol A and B for tryptic digest etc of
> human
> > serum samples. In the same final elution volume (spin tip cleanup),
> > protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
> > for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
> > mode. We identify similar numbers of protein groups for protocol A and B,
> > BUT protocol B identifies more peptides and precursors.
> >
> > It seems like protocol A retrieves more peptides, but fewer *different*
> > peptides, i.e. it might just digest the highly abundant proteins such as
> > albumin more than protocol B?
> >
> > Both protocols A and B use a simple workflow of denaturation, alkylation,
> > tryptic digest and cleanup.
> >
> > 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
> > pronouns: she/her/hers
> >
> > https://vogellab.bio.nyu.edu/<https://vogellab.bio.nyu.edu> ---
> @CVogelNYC
> >
> > “If your dreams don't scare you, they are not big enough” Ellen Johnson
> > Sirleaf
> >
> > ――
> > View topic
> >
>
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> > Leave group <email obscured>?Subject=Unsubscribe
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> > Leave group <email obscured>?Subject=Unsubscribe
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> >
>
> --
>
> 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
> pronouns: she/her/hers
>
> https://vogellab.bio.nyu.edu/<https://vogellab.bio.nyu.edu/> ---
> @CVogelNYC
>
> “If your dreams don't scare you, they are not big enough” Ellen Johnson
> Sirleaf
>
> ――
> View topic
>
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>
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>
--
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
pronouns: she/her/hers
https://vogellab.bio.nyu.edu/ --- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
Sirleaf
Hi Christine,
Given that you are seeing similar numbers of proteins identified in both preps,
but more peptides in B, is it possible that you are getting a difference in
trypsin digestion efficiency such that B gives you a mixture of fully cleaved
and missed cleaved peptides? Or alternatively, could protocol B be inducing
more artefactual modifications (deamidation, oxidation, pyro-glu formations)
than protocol A?
Best regards,
Martin
Martin Middleditch
Lead Technologist (Mass Spectrometry)
School of Biological Sciences
University of Auckland
Room 301-422
23 Symonds Street
Auckland
(09) 923-7682 or 923-7540
Rest of post
________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Christine Vogel
<email obscured>>
Sent: Friday, January 29, 2021 8:56 AM
To: <email obscured>; Smruti D Pushalkar
Subject: Re: [ABRF Discussion Forum] serum samples -- peptide quantification
vs. identification in the MS
We used the Pierce Fluorometric Peptide Assay to estimate peptide
concentration (labels N-terminal amines). That's another source for bias.
TICs are higher for protocol B (the one with lower measured peptide
concentration but higher identification in MS).
I am tempted to go ahead with protocol B, but still puzzled about the
higher apparent peptide concentration in protocol A.
Best,
Christine
On Thu, Jan 28, 2021 at 2:50 PM Anderson, David Eric (NIH/NIDDK) [E] <
<email obscured>> wrote:
> Is your assumption about the difference in concentration of the material
> born out in the TIC's. Does the overall look of the raw data look likes it
> comes from the same amount of peptides?
>
> D. Eric Anderson, Ph.D
> Mass Spectrometry Facility, NIDDK
> NIH
>
>
> Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
> (301)-496-7546
>
>
>
> ________________________________________
> From: Christine Vogel <email obscured>>
> Sent: 28 January 2021 14:04
> To: <email obscured>; Smruti D Pushalkar
> Subject: [ABRF Discussion Forum] serum samples -- peptide quantification
> vs. identification in the MS
>
> Dear Colleagues,
>
> I'm hoping to pick your brain on something that puzzles me: despite lower
> overall peptide yield in one prep of serum samples vs. a different prep,
> we observe higher identification of peptides in mass spec analysis.
>
> Long story: we are testing protocol A and B for tryptic digest etc of human
> serum samples. In the same final elution volume (spin tip cleanup),
> protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
> for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
> mode. We identify similar numbers of protein groups for protocol A and B,
> BUT protocol B identifies more peptides and precursors.
>
> It seems like protocol A retrieves more peptides, but fewer *different*
> peptides, i.e. it might just digest the highly abundant proteins such as
> albumin more than protocol B?
>
> Both protocols A and B use a simple workflow of denaturation, alkylation,
> tryptic digest and cleanup.
>
> 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
> pronouns: she/her/hers
>
> https://vogellab.bio.nyu.edu/<https://vogellab.bio.nyu.edu> --- @CVogelNYC
>
> “If your dreams don't scare you, they are not big enough” Ellen Johnson
> Sirleaf
>
> ――
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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
pronouns: she/her/hers
https://vogellab.bio.nyu.edu/<https://vogellab.bio.nyu.edu/> --- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
Sirleaf
――
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We used the Pierce Fluorometric Peptide Assay to estimate peptide
concentration (labels N-terminal amines). That's another source for bias.
TICs are higher for protocol B (the one with lower measured peptide
concentration but higher identification in MS).
I am tempted to go ahead with protocol B, but still puzzled about the
higher apparent peptide concentration in protocol A.
Best,
Christine
On Thu, Jan 28, 2021 at 2:50 PM Anderson, David Eric (NIH/NIDDK) [E] <
Rest of post
<email obscured>> wrote:
> Is your assumption about the difference in concentration of the material
> born out in the TIC's. Does the overall look of the raw data look likes it
> comes from the same amount of peptides?
>
> D. Eric Anderson, Ph.D
> Mass Spectrometry Facility, NIDDK
> NIH
>
>
> Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
> (301)-496-7546
>
>
>
> ________________________________________
> From: Christine Vogel <email obscured>>
> Sent: 28 January 2021 14:04
> To: <email obscured>; Smruti D Pushalkar
> Subject: [ABRF Discussion Forum] serum samples -- peptide quantification
> vs. identification in the MS
>
> Dear Colleagues,
>
> I'm hoping to pick your brain on something that puzzles me: despite lower
> overall peptide yield in one prep of serum samples vs. a different prep,
> we observe higher identification of peptides in mass spec analysis.
>
> Long story: we are testing protocol A and B for tryptic digest etc of human
> serum samples. In the same final elution volume (spin tip cleanup),
> protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
> for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
> mode. We identify similar numbers of protein groups for protocol A and B,
> BUT protocol B identifies more peptides and precursors.
>
> It seems like protocol A retrieves more peptides, but fewer *different*
> peptides, i.e. it might just digest the highly abundant proteins such as
> albumin more than protocol B?
>
> Both protocols A and B use a simple workflow of denaturation, alkylation,
> tryptic digest and cleanup.
>
> 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
> pronouns: she/her/hers
>
> https://vogellab.bio.nyu.edu/ --- @CVogelNYC
>
> “If your dreams don't scare you, they are not big enough” Ellen Johnson
> Sirleaf
>
> ――
> View topic
>
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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
pronouns: she/her/hers
https://vogellab.bio.nyu.edu/ --- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
Sirleaf
Is your assumption about the difference in concentration of the material born
out in the TIC's. Does the overall look of the raw data look likes it comes
from the same amount of peptides?
D. Eric Anderson, Ph.D
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: 28 January 2021 14:04
To: <email obscured>; Smruti D Pushalkar
Subject: [ABRF Discussion Forum] serum samples -- peptide quantification vs.
identification in the MS
Dear Colleagues,
I'm hoping to pick your brain on something that puzzles me: despite lower
overall peptide yield in one prep of serum samples vs. a different prep,
we observe higher identification of peptides in mass spec analysis.
Long story: we are testing protocol A and B for tryptic digest etc of human
serum samples. In the same final elution volume (spin tip cleanup),
protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
mode. We identify similar numbers of protein groups for protocol A and B,
BUT protocol B identifies more peptides and precursors.
It seems like protocol A retrieves more peptides, but fewer *different*
peptides, i.e. it might just digest the highly abundant proteins such as
albumin more than protocol B?
Both protocols A and B use a simple workflow of denaturation, alkylation,
tryptic digest and cleanup.
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
pronouns: she/her/hers
https://vogellab.bio.nyu.edu/ --- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
Sirleaf
――
View topic http://list.abrf.org/r/topic/2QblHaTaOsQtmYaccziECV
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Electrophoretic Mass Spectrometry Technology Corporation (EMAST) has an
immediate opening for an Instrument Technician (permanent, full time with
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The successful candidate will be involved in the beta prototype and
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For additional information or to submit a resume, please contact me directly at
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feel free to forward this email to any colleagues you think may be interested.
Best Regards,
Dear Colleagues,
I'm hoping to pick your brain on something that puzzles me: despite lower
overall peptide yield in one prep of serum samples vs. a different prep,
we observe higher identification of peptides in mass spec analysis.
Long story: we are testing protocol A and B for tryptic digest etc of human
serum samples. In the same final elution volume (spin tip cleanup),
protocol A yields 700 ng/ul, while protocol B yields 350 ng/ul. Adjusting
for this, we inject 350 ng each for analysis on an q-Exactive HF in DIA
mode. We identify similar numbers of protein groups for protocol A and B,
BUT protocol B identifies more peptides and precursors.
It seems like protocol A retrieves more peptides, but fewer *different*
peptides, i.e. it might just digest the highly abundant proteins such as
albumin more than protocol B?
Both protocols A and B use a simple workflow of denaturation, alkylation,
tryptic digest and cleanup.
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
pronouns: she/her/hers
https://vogellab.bio.nyu.edu/ --- @CVogelNYC
“If your dreams don't scare you, they are not big enough” Ellen Johnson
Sirleaf