Posts in ABRF Discussion Forum

All -
I'm looking for various metrics - beyond the obvious
revenue/expense/subsidy/customer base -  info on how other Institutions review
Core Facilities.   Are there metrics that are being used that will allow
comparisons across many different types of Cores such as Flow vs an MRI Core?

Thank you!


Susanna Perkins
Director, Research Cores & Operations
Research Core Administration, S2-106
Tel: (508) 856-8255
Fax: (508) 856-2303
E-Mail:  <email obscured><email obscured>>

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Hi Andor,

Although I haven't used them, a colleague recommended these folks to me:

https://seqgen.com/


Caveats and disclaimers...

Cheers!

Steve


On Mon, Sep 16, 2019 at 2:55 PM Andor J Kiss <email obscured>> wrote:

> Hello,
>
>      Are there any alternatives to ABI (LT/Thermo Fisher) for service
> contracts for the ABI 3130/xl/ and ABI 3730 instruments here in the US?
> Looking for recommendations.
>
> Thanks,
> --
>
> _____________________________________________________________
> Andor J Kiss, PhD
> Director - Center for Bioinformatics & Functional Genomics
> 086 Pearson Hall - Miami University
> 700 East High Street, Oxford
> Ohio 45056
> USA
>
> eMAIL: <email obscured> <email obscured>>
> Telephone: +1 (513) 529-4280
> Fax: +1 (513) 529-2431
> JAMI / SKYPE: andorjkiss <https://jami.net/download/>
>
> URL (CBFG): http://miamioh.edu/cas/academics/centers/cbfg/
> URL (CBFG Services):
>
>
https://www.scienceexchange.com/labs/center-for-bioinformatics-functional-genomics
>
> URL (Research): http://openwetware.org/wiki/User:Andor_J_Kiss
>
>
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--

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/

Dear Manfred,

Thank you so much for your help!

Kind regards,

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
Manfred@newtoncon
Sent: Thursday, August 29, 2019 1:50 AM
To: <email obscured>
Subject: [External] Re: [ABRF Discussion Forum] dried blood spot proteomics

Hi,
look for papers from Chris Borchers' group. They have done this from dired
blood spots Manfred

Am 28.08.19, 20:09 schrieb "ABRF Discussion Forum im Auftrag von Ding, Hua"
<email obscured> im Auftrag von <email obscured>>:

    Hi,

    I need help to identify smaller proteins from the dried blood spot filter
paper via LCMS.  Does anyone having good experience for dried blood spots
proteomics workflow?

    Thank you so much in advance for your advice.

    Best,

    Hua

    Hua Ding,  Ph.D.
    Research Associate
    Protein and Proteomics Core Facility
    The Children's Hospital of Philadelphia

    <email obscured><email obscured>>
    Phone:  267-426-5552


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Skyline (https://skyline.ms) should be a nice way to find any set of compounds
or defined masses in any number of chromatograms, in a vendor neutral and
shareable format.

Cheers

Will

-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Margaret Coe
Sent: Thursday, August 29, 2019 4:52 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] A question about data analysis on Thermo
software

Hi Kym,
You could you use TraceFinder (or the older ExactFinder).  If you use
chromatogram 2 as the background to be subtracted, you will get the
differences.  You'd need to do this for both the #2 and #3 chromatograms and
then compare the resulting list.  You can do this with Compound Discoverer,
too, but I'm pretty sure it can only have 1 file as the background, as well.

If this is one-off case, you could get in the software on a trial basis for 1
or 3 months, whatever they do.


Best regards,
Margaret Coe
Senior Research Technician
Equity Committee Chair
Liggins Institute

DDI +64 9 923 3432 Ext 83432 E <email obscured> W liggins.auckland.ac.nz A
The University of Auckland, Building 503, Level 2, 85 Park Road, Auckland P
Private Bag 92019, Auckland 1142, NZ





-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Kym Faull
Sent: Friday, 30 August 2019 5:00 AM
To: <email obscured>
Cc: Li, Shen <email obscured>>
Subject: [ABRF Discussion Forum] A question about data analysis on Thermo
software

― 1 file link ―

We have three LC/MS chromatograms collected on a QE instrument.  We are looking
for ions in Chromatogram 1 that are not present in either Chromatograms 2 and
3.  Other than manually interrogating the data sets is there some more
convenient/automated  approach to 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, 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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SIEVE from Thermo had a function like this. But I don't know how well it
works. Thermo stopped supporting it lately, but it's probably worth trying.
'

Yan

On Thu, Aug 29, 2019 at 1:00 PM Kym Faull <email obscured>> wrote:

> ― 1 file link ―
>
> We have three LC/MS chromatograms collected on a QE instrument.  We are
> looking for ions in Chromatogram 1 that are not present in either
> Chromatograms 2 and 3.  Other than manually interrogating the data sets is
> there some more convenient/automated  approach to 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, 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>>
>
>
>
>
>
>
>
>
> ― 1 file ―
>
>   □ smime.p7s  (1.2kb)
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>
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>

Hi,
look for papers from Chris Borchers' group. They have done this from dired
blood spots
Manfred

Am 28.08.19, 20:09 schrieb "ABRF Discussion Forum im Auftrag von Ding, Hua"
<email obscured> im Auftrag von <email obscured>>:

    Hi,

    I need help to identify smaller proteins from the dried blood spot filter
paper via LCMS.  Does anyone having good experience for dried blood spots
proteomics workflow?

    Thank you so much in advance for your advice.

    Best,

    Hua

    Hua Ding,  Ph.D.
    Research Associate
    Protein and Proteomics Core Facility
    The Children's Hospital of Philadelphia

    <email obscured><email obscured>>
    Phone:  267-426-5552


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Hi,

I need help to identify smaller proteins from the dried blood spot filter paper
via LCMS.  Does anyone having good experience for dried blood spots proteomics
workflow?

Thank you so much in advance for your advice.

Best,

Hua

Hua Ding,  Ph.D.
Research Associate
Protein and Proteomics Core Facility
The Children's Hospital of Philadelphia

<email obscured><email obscured>>
Phone:  267-426-5552

We've used it here in manchester.
It's an interesting bit of kit.
Different to the off gel system, in that while both will do protein
fractionation, gel free is compatible with SDS.
Never tried other detergents
Resolution not great, but good enough I guess
Requires a LOT of sample for loading.
Ronan

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 ˚C EtOH and washed twice with -20 ˚C
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

Am 20.08.19, 13:42 schrieb "ABRF Discussion Forum im Auftrag von Hana Konečná"
<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 – Proteomics
    Masaryk University Campus, Building A26
    Kamenice 5, 625 00 Brno
    Czech Republic

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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

Am 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
    >
    >
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    --

    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/

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