Posts in ABRF Discussion Forum

I want to use publically available tandem mass spectrometry data to search for
the presence of a hypothetical protein that is not in a protein database.

Specifically, I am looking for mouse brain tandem mass spectrometry data from a
deep proteome study or several analyses covering different mouse brain regions.

Can someone recommend a good source for such data?
What search strategy and engine would be good to use?

Chris

Dear Andy,
the „Viper“ Valve is just a normal Valco valve. You can use the normal Valco
fittings e.g. these: https://vici.com/vfit/fs_intro.php
<https://vici.com/vfit/fs_intro.php> or a long nut such as LZN1-10 or XZLN1-10
and a longer PEEK sleeve. Just use a 360µm ID silica capillary as a connector
between the valve and the columns’ union. One can also use connecting
capillaries of the trap column (the one with the cartridge) and connect using
this short piece with Vipers on both sides.
Success!
Goran



> Am 05.07.2019 um 19:54 schrieb <email obscured>> <email obscured>>:
>
> A customer of ours is buying a capillary that terminates in the 1/16" female
unions that we routinely use as end fittings.  He's just asked the following
question:
> *********************************************
> For we are using an UltiMate 3000 nLC (Thermo Fisher Scientific), the columns
have to be connected to the valves through Viper.
>
> So far, we have been using columns manufactured by Thermo and they are
pre-assembled with the NanoViper.
>
> Once we tried using a capillary column without Viper, we have to stuck it to
the valve with a PEEK sleeve. It was extremely cumbersome and we end up
breaking the column.
>
> Do you have any advice on connecting the capillary columns to Thermo’s Viper
valve?

> *********************************************
> Does anyone have any advice to offer in this regard?
>
> Andy Alpert
> PolyLC Inc.
> <email obscured>
>
>
> ――
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>

Goran Mitulović, Ph.D.
Head Proteomics Core Facility
Medical University of Vienna
Clinical Institute of Laboratory Medicine and Proteomics Core Facility
Waehringer Guertel 18-20,
Ebene 5H, LS5H
A-1090 Wien, Austria
Tel: +43 1 40 400 53750
Email: <email obscured>
Web: www.meduniwien.ac.at/cf-proteomics

Hi Angel,
Dirk is right in stressing your attention that the cited paper describes the
preparation of polyclonal, but not monoclonal, antibodies. As for monoclonal
antibodies, you can get several hybridoma clones that produce mAbs with
differing specificities for each of the peptide used. Are you planning to use
one immunization instead of three? If so, you can achieve the goal. You can
also prepare one carrier with three peptide haptens, if you use different
linkages (NH2- to NH2, NH2 to SH, to diazo-Tyr). But it is possible if the
previously conjugated peptides do not contain side-chain groups that are
susceptible to the latter used linkage reagents.

Dear ARG Members,

I have a question about using peptide pools conjugated to carrier protein for
immunization. I have an investigator who wants to produce a monoclonal antibody
targeting a specific histidine residue in/near the active site of a particular
membrane protein to block enzymatic activity.

The PI identified 3 peptides that were predicted to be immunogenic.  Rather
than pick one, I suggested using all three peptides. Also, I suggested that
equimolar amounts of the three peptides could be conjugated to the same carrier
molecule, since the peptides are identical except 3-4 amino acid differences. I
spoke with a technical rep from Thermo Scientific, and this person assured me
that this is a common practice. However, I have only been able to find one
example of a somewhat similar approach in the literature
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015836/). Is this approach
commonly used? Do you know of a publication that has used peptide pools to
produce site directed antibodies?

Thank you for your advice!

Angel


Edith “Angel” Sampson, Ph.D.
Monoclonal Antibody Scientific Director

UF ICBR
2033 Mowry Road, Gainesville, FL 32610
Biotech.ufl.edu | Tel: 352.273.8039| Fax: 352.273.8070

Since I'm also interested in this topic, I'll throw in my 2 cents to get
the discussion going.

My understanding is there are concerns with losing proteins that are bound
to major proteins when using depletion, plus cost is not trivial. So people
would want to stay away from it if they can. With a good instrument, you
probably can get >500 protein IDs with 2-3 hour analysis. Then you can get
more if you fractionate. It takes more instrument time, but depending on
how expensive instrument time is for you, you might find it worth it.

Then the last time I looked into depletion, someone recommended ProteoMiner
by BioRad,
http://www.bio-rad.com/en-us/sku/1633006-proteominer-protein-enrichment-small-capacity-kit?ID=1633006
.
I never got to try it as the project already moved on by the time I learned
about it and would love to hear feedback from folks.

Yan

On Wed, Jun 26, 2019 at 2:42 AM turck <email obscured>> wrote:

> I am sure this has been a topic previously. But things may have changed
> over time ...
>
> I need recommenations for removing abundant proteins from plasma and CSF.
>
> - what are the best kits?
> - expensive vs. cheap hits, how big is the difference in performance (is
> it worth spending more money?)?
> - will the available kits work with macaque specimens (blood, CSF)?
>
> I have seen a publication on qantitative mass spec of plasma proteins that
> did not use removal of abundant proteins. Can this be done on a routine
> basis?
>
> Thanks much!
>
> Chris
> ――
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>
>

Dear all
A colleague has been thinking on analysing samples containing saponins.
Does anyone know if these products are mass-spec friendly? The glycoside
moieties of it may be okay (I guess) but I've heard of the detergent property
of saponins so that I'm affraid that it may clog our pre-and analytical C18
columns, or even, enter our mass spec and dirty the Q0, Q1 and so on. Any
suggestion/critics/comments?

Leo

Thank you Brian and Joerg for your feed back!!
Dr. Carlos H. Paván
Director Técnico Laboratorio Nacional de Investigación y Servicios en Péptidos
y Proteínas. Estudios por Espectrometría de Masas.LANAIS-PROEM. UBA
CONICET.Paraguay 2155 6to Piso.
 Buenos Aires, Argentina.

    El viernes, 21 de junio de 2019 12:47:05 p. m. GMT-3, Brian Hampton
<email obscured>> escribió:

 Hi Joerg,

You may want to consider the cost of maintaining a water purification system
compared to purchasing pure water.  An MS core facility near me uses Thermo
Scientific NERL grade water in 5 gallon packs because it is so much less
expensive than maintaining a water purification unit.  Since it is in some kind
of plastic (probably low density polyethylene) pack contained by an outer
cardboard box with a built in valve to access the water,  I asked if they see
plasticizer ion signals, and they do but only in GCMS.  In most of their use
cases, sodium leaching from glass is more of a problem.

At the price point in the US, I would have to use about 175-200 gallons of this
NERL water to equal the cost of replacing the cartridges and filters in a
Millipore water purification unit that takes DI water as input.  If your system
has to start from unpure water then maintenance costs will be even higher.
Shipping costs of water to your location could be another cost to consider.

Here is the URL for the water I am referring to:
https://www.fishersci.com/shop/products/nerl-reagent-grade-water/23249581

Regards,

Brian


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Hi,
We typically monitor the TOC (Trace organic compounds). A high-energy UV-lamp
is used to break down larger organic molecules.
Joerg

Von meinem iPhone gesendet

> Am 21.06.2019 um 15:08 schrieb Carlos H. Pavn
<email obscured>>:
>
> Dear Mass Spec colleagueswhat parameter do you usually check for Milli Q
water for MS, apart from conductance? we are setting up uor lab for a 4500
QTRAP for protein analysis.
> Some guy told me about a carbon control but I don't I don't remember,
>
> Best regards
> Dr. Carlos H. Pavn
> Director Tcnico Laboratorio Nacional de Investigacin y Servicios en Pptidos y
Protenas. Estudios por Espectrometra de Masas.LANAIS-PROEM. UBA
CONICET.Paraguay 2155 6to Piso.

> Buenos Aires, Argentina.
> DD
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>

Hi All,

We are in the midst of a project extracting RNA from FFPE curls from brain
tissue using the Beckman FormaPure Total XL kit. We are able to obtain good
yields of RNA with near zero DNA contamination as determined by qubit. However,
the fragment analyzer traces show a single mass of HMW RNA migrating at
~6000nt. I am thinking that this might be the result of some contaminant
preventing efficient migration of the RNA, but im not certain. Traces are
attached here for reference.

Surprisingly, Beckman says they have seen this too and to not be concerned, but
we're not going to make libraries or run Nanostring on such samples if we can't
properly QC them up front. Interestingly, we have not seen these problems with
the older FormaPure Total kits.

If anyone has seen something similar and has either 1) successfully diagnosed
the issue or 2)has used this material successfully in NGS or related
applocatios that would be great to know.

Thank you all as always for your valuable insights!

Best,
Fred

Sent from my mobile device. Please excuse the brevity and typos.