Posts in ABRF Discussion Forum

Hello,

I am curious if anyone has attempted Slide-seq in their labs. The method seems
pretty straightforward except for the sequencing of the pucks, which seems
laborious. I would like to hear from folks who have tried this method and I
would love to follow-up with you if you are attending the ABRF meeting in San
Antonio.

Paper: https://www.biorxiv.org/content/10.1101/563395v1

Best,

Anoja

out of interest, is it possible to run an I-Class and M-Class from the same
PC with this configuration?  I would assume not, as in another setup we
have (Waters Synapt G2Si) there is a lot of deconfiguration /
recongiuration that needs to happen when switching between I- and M-class
inlets.  So I assume that there will be the same issues via LC Devices for
a Thermo MS?


thanks
Tony


On Wed, 13 Mar 2019 at 20:16, Roy_Martin <email obscured>> wrote:

> ― 1 file link ―
>
> Adam,
> Took a bit of digging but here's a more or less official comment.
>
> There is an official version of the software for the M-Class in a Thermo
> Excalibur installation.
>
>
http://www.waters.com/waters/support.htm?locale=en_US&lid=134899891&cid=511442&type=DWNL

>
> This might be different from the version that comes with the instrument.
> I've also included installation instructions in the pdf file in the link.
> This version of software hasn't yet been tested for Win10.
>
> Please do log a call with Field Service to get help if you continue to
> need help
>
> Roy
>
> (be with all the cool kids in San Antonio soon!)
> ― 1 file ―
>
>   □ 715005252 MClass Excalibur.pdf  (4mb)
>
> http://list.abrf.org/r/file/mEhf9VegFVeSokXwDEPFxQcgFiw-fj1U-2JsIARe/
>
> ――
> View topic http://list.abrf.org/r/topic/4nsn10L0sUFryJzRFA7sU2
> Leave group <email obscured>?Subject=Unsubscribe
>
>

--
Dr. Tony R. Larson
Head of Metabolomics & Proteomics, Department of Biology, Area 15
University of York
Wentworth Way
Heslington
York YO10 5DD
UK

Tel: +44(0)1904 328 733 (office)
Tel: +44(0)7833 471 685 (mobile)

<email obscured>
http://scholar.google.com/citations?user=9hLFka4AAAAJ
www.york.ac.uk/biology/technology-facility/proteomics/
www.york.ac.uk/mass-spectrometry

Hi Adam,

The version of LC Devices needed depends on the version of Thermo's Foundation
and Xcalibur that your instrument is using. I am running different versions on
different instruments, but the M-Class should start with 1.55 drivers,
regardless. Firmwares that work for my M-Class BSM is version 1.56.154, SM
1.56.376 and TVM 1.53.25. The standard way is to install the Foundation,
Xcalibur, LC Devices and then the M-Class drivers combined with Acquity UPLC
Console. However, a few years ago I had to install the M-Class drivers combined
with Acquity UPLC Standalone Console first to make sure that I could control
the LC then install the Thermo software in after that.

If you have several versions of LC Devices installed on the same PC already,
you might have to uninstall them, each time rebooting the PC and then
physically remove the leftover files from the PC's registry. The leftover files
confuse the Xcalibur and the defective/partially uninstalled LC Devices
software may be mistakenly loaded by Xcalibur.

I have had to wipe the hard drive and started to from scratch before and that
was much faster than the troubleshooting to have the systems working smoothly.
While at it, check to see that you have Windows updated to the most current dot
NET Framework, either 4.6 or 4.7.2.

Good luck.

Kwasi



-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of adam.dowle
Sent: Tuesday, March 5, 2019 11:04 AM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Advice on configuring an Orbitrap Fusion
with a Waters mClass UPLC

Hello Kwasi,

Many thanks for your reply.  I agree re firmware miscommunication issue but I
am not sure how to sort it. Which version of the drivers are your using?  LC
Devices contains 1.50, we also have 1.53 and 1.55 on disk, while 1.56 can be
downloaded from Waters.  Do you apply the firmware from the loader before or
after installing through LC Devices? LC Devices gives the option to either
install 1.50 or not so either way is possible.

Thanks for your help,

Adam
――
View topic http://list.abrf.org/r/topic/7v7l6jhNL50Me4sizS1W96
Leave group <email obscured>?Subject=Unsubscribe


________________________________
The materials in this message are private and may contain Protected Healthcare
Information or other information of a sensitive nature. If you are not the
intended recipient, be advised that any unauthorized use, disclosure, copying
or the taking of any action in reliance on the contents of this information is
strictly prohibited. If you have received this email in error, please
immediately notify the sender via telephone or return mail.

Hi Adam,
This sounds a lot like what we went through some years ago to get an
Orbi-XL to control an Agilent 1100 system.  HUUUGE pain!  We got it to work
by making sure ALL of the LC components had the specified firmware versions
(not close to the latest ones), and the specified Xcalibur version.  It
eventually worked, but was not too stable.  Once running it was OK, but it
often took several reboots if the computer had to go down for any reason,
so we would avoid reboots like the plague.  Good luck!

Regards,
David Hawke

On Tue, Mar 5, 2019 at 11:03 AM adam.dowle <

<email obscured>> wrote:

> Hello Kwasi,
>
> Many thanks for your reply.  I agree re firmware miscommunication issue
> but I am not sure how to sort it. Which version of the drivers are your
> using?  LC Devices contains 1.50, we also have 1.53 and 1.55 on disk, while
> 1.56 can be downloaded from Waters.  Do you apply the firmware from the
> loader before or after installing through LC Devices? LC Devices gives the
> option to either install 1.50 or not so either way is possible.
>
> Thanks for your help,
>
> Adam
> ――
> View topic http://list.abrf.org/r/topic/7v7l6jhNL50Me4sizS1W96
> Leave group <email obscured>?Subject=Unsubscribe
>
>

Hello Kwasi,

Many thanks for your reply.  I agree re firmware miscommunication issue but I
am not sure how to sort it. Which version of the drivers are your using?  LC
Devices contains 1.50, we also have 1.53 and 1.55 on disk, while 1.56 can be
downloaded from Waters.  Do you apply the firmware from the loader before or
after installing through LC Devices? LC Devices gives the option to either
install 1.50 or not so either way is possible.

Thanks for your help,

Adam

Hello,

Is anyone out there running an Orbitrap Fusion or similar with a Waters mClass
UPLC?  If so do you have any tips on how to make the software communication
stable?  We have recently set this configuration up ourselves using LC Devices
3.2, with Xcalibur 4.2 and Tune 3.1.  The mClass can be controlled through
Xcalibur but we see frequent stoppages in sequence runs.  Sometimes the
sequence just sits on pause part way through for no clear reason and will
resume when we hit play.  Other times we get an AcquityServer stopped error,
which requires a PC restart to get it going again.  I understand that the
mClass is not a Thermo supported device in the latest Thermo software versions
of Xcalibur, Sii etc. but if anyone has got this going successfully I would be
very grateful for advice to help me do the same.

Many thanks,

Adam

Hello, in our lab we perform genetic tests for animals. Does anybody has some
experience  about room temperature blood banking ( liofilized ir using  some
buffer??)??i am interested in share some tips
Than you
Vincenzo Landi

Enviado desde mi iPhone

El 21 feb. 2019, a la(s) 23:35, Noah D4 <email obscured>> escribió:

> I was about to embark on a project using Asp-N but I'm now reconsidering
after discovering how expensive this stuff is. Looks like it costs 10x or more
than other common enzymes. I see a few places that will make recombinant on a
0.5-2mg scale for $1000-2000, which seems like a great deal if the product
works satisfactorily.

>
> Anyone with Asp-N experience have any advice?
>
> Thanks,
>
> Noah
>
> Noah Dephoure, Ph.D.
> Assistant Professor
> Sandra and Edward Meyer Cancer Center
> Department of Biochemistry
> Weill Cornell Medical College
> 413 East 69th Street, BRB1520
> ――
> View topic http://list.abrf.org/r/topic/4FooDQQ85RQgJoHlWSOdXE
> Leave group <email obscured>?Subject=Unsubscribe
>

Hi Goran,
I worked on peptides from human hemofiltrate - some years back. In this time,
the few I run on a LCQ I just searched without specific digestion against the
normal protein database using SEQUEST and got some hits. It is a kind of
middle-up approach, larger protein fragments. Do you have an idea about the
average size of the peptides?
In my experience I would use PEAKS software to search as this allows the
non-specific digestion quite well, tried some ant venom peptides about a year
back. Of course, ants are not well sequenced, but at first I got some de-novo
sequences and some sequences from other species. At least the de-novo could be
verified manually in many cases. The software can be fully tried for 2 weeks.
Manfred

Am 25.02.19, 12:01 schrieb "ABRF Discussion Forum im Auftrag von Goran
Mitulović" <email obscured> im Auftrag von
<email obscured>>:

    Dear All,

    I am trying to identify endogenous peptides extracted from a tissue - no
    digest performed- upon nanoLC-MS/MS. Can somebody help with directing me
    to a usable peptide database?

    Many thanks

    Goran



    ――
    View topic http://list.abrf.org/r/topic/6gw93AGMguj5znbTvDy2xF
    Leave group <email obscured>?Subject=Unsubscribe

Brian,

This looks promising, thanks. I'm curious how it would work for mg's of whole
cell extract. Seems simple enough to try.

Noah

Hi Noah,

I used it recently to do a "double digest" for a project where a very long
tryptic peptide was of interest.  The Asp-N digest (used 100ng per sample)
was on the already generated tryptic peptides.  In 4 hours, I obtained the
desired shorter peptide..
I agree with you that it is very expensive ( we paid over $180.00) for a
2ug vial.  However, for special projects, it is certainly worth it.
I also do not know how it would perform as the first enzyme of choice.
Tend to use Asp-N and Glu-C to carry out "double digests" on samples which
already has Trypsin protease as the primary enzyme.

Best,
Hediye.

On Thu, Feb 21, 2019 at 5:35 PM Noah D4 <email obscured>> wrote:

> I was about to embark on a project using Asp-N but I'm now reconsidering
> after discovering how expensive this stuff is. Looks like it costs 10x or
> more than other common enzymes. I see a few places that will make
> recombinant on a 0.5-2mg scale for $1000-2000, which seems like a great
> deal if the product works satisfactorily.
>
> Anyone with Asp-N experience have any advice?
>
> Thanks,
>
> Noah
>
> Noah Dephoure, Ph.D.
> Assistant Professor
> Sandra and Edward Meyer Cancer Center
> Department of Biochemistry
> Weill Cornell Medical College
> 413 East 69th Street, BRB1520
> ――
> View topic http://list.abrf.org/r/topic/4FooDQQ85RQgJoHlWSOdXE
> Leave group <email obscured>?Subject=Unsubscribe
>
>