________________________________________
From: Anderson, David Eric (NIH/NIDDK) [E]
<email obscured>>
Sent: 12 November 2020 10:16
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] mass spec peptide isotopologue pattern
intensity differences
But do you see effects like this in SILAC heavy vs. light? Do the N15
containing SIALC AA have shifts from the Light channels?
It seems like it is tied up in the direct H-bonding so the effect must be
greater. Most of the D effects we see are for less polarized C-D vs C-H bonds..
Just trying to understand the magnitude. I know this stuff is additive from my
experience with DM labeling so I think it adds up because there are so many
sites.
D. Eric Anderson
Mass Spectrometry Facility, NIDDK
NIH
Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
(301)-496-7546
________________________________________
From: Goran Mitulović <email obscured>>
Sent: 12 November 2020 10:08
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] mass spec peptide isotopologue pattern
intensity differences
Sorry! Mea culpa! I was referring to the heav isotope. D has, however,
an even larder effect.
Best,
Goran
On 12.11.2020 16:04, Anderson, David Eric (NIH/NIDDK) [E] wrote:
> It is not D it is N15. I have never seen such a dramatic effect for silac (up
to 10 x n15 c13) - it seems like it could add up because these are uniform N15
labeled and they are involved directly in real hydrogen bonds.
>
> D. Eric Anderson
> Mass Spectrometry Facility, NIDDK
> NIH
>
>
> Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
> (301)-496-7546
>
>
>
> ________________________________________
> From: Goran Mitulović <email obscured>>
> Sent: 12 November 2020 09:59
> To: <email obscured>
> Subject: Re: [ABRF Discussion Forum] mass spec peptide isotopologue pattern
intensity differences
>
> Actually, not weird at all. The isotopic effect of D on chromatographic
> behavior has been described earlier and it is the explanation for Chris'
> observation. One can increse the effect by modifying the loading and the
> eluting mobile phases. Add a certain % of methanol to the eluting mobile
> phase and it will become more distinct.
>
> Best,
>
> Goran
>
>
> On 12.11.2020 15:41, Anderson, David Eric (NIH/NIDDK) [E] wrote:
>> That is weird and I guess Steve is right on but it is such a large effect
and is sort of worrisome.
>>
>> You actually have a nice population there and you can probably graph the
peak time for each number of N15's.
>>
>> Since there are a lot of sites and almost everyone potentially makes a
hydrogen bond that can add up. We see big effects from deuterium in SILAC
(lysine D4) and dimethyl labeling (in nether case do the D's make normal
hydrogen bonds though C-H bonds are a bit polarized)
>>
>> My general view is that the effect should be smaller for N-14 to N-15 and
C-12 to C-13 because the mass difference of the nucleus is smaller. But
13x(1/14) is almost 1 so in silly math this is like having 2 H-1 to H-2 (if the
effect were just linear with mass difference which isn't true) and we would be
able to see different retention times for these forms (but normally just
barely).
>>
>> It could be somewhat specific to the peptide if it is partially structured
under these conditions. If you are not heating your column this may improve
co-elution but that is just a guess.
>>
>> All the best,
>>
>>
>> Eric
>>
>>
>> D. Eric Anderson
>> Mass Spectrometry Facility, NIDDK
>> NIH
>>
>>
>> Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
>> (301)-496-7546
>>
>>
>>
>>
>> ________________________________________
>> From: Chris Turck <email obscured>>
>> Sent: 12 November 2020 09:17
>> To: Steve Eyles; <email obscured>
>> Subject: Re: [ABRF Discussion Forum] mass spec peptide isotopologue pattern
intensity differences
>>
>> Hi Steve, right on! I checked the chromatogram and it looks as though the
15N-labeld peptide indeed elutes a little earlier than the non-labeled peptide.
A neutron can make a difference! Thanks for your quick response! Chris
>>
>>
>> From: Steve Eyles <email obscured>>
>> Sent: Donnerstag, 12. November 2020 14:42
>> To: <email obscured>
>> Cc: Chris Turck <email obscured>>
>> Subject: Re: [ABRF Discussion Forum] mass spec peptide isotopologue pattern
intensity differences
>>
>> Hi Chris
>>
>> I don’t think it’s unusual for there to be a very slight difference in
elution times between non-labeled and isotopically labeled peptides. 15N
labeling changes mass but also can affect bond strengths/H-bonding, etc.
>> I’ve seen some, but not all, peptides exhibiting slight shifts in elution
time. Try plotting the XIC/EIC of single isotopologue masses that would be
unique to the labeled and unlabeled peptide and I would guess you’ll see a
shift in elution time of the peak max.
>>
>> 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 Nov 12, 2020, at 8:28 AM, turck
<email obscured><email obscured>>> wrote:
>>
>> ― 1 file link ―
>>
>> In the attachment you see two MS1 scans from a single LC-MS/MS run with
isotopologue patterns of the same peptide in its non-labeled form (blue bar)
und 15N-labeled form (~ 90%, red bar).
>> I am a bit puzzled by the difference in isotopologue signal intensities of
the non-labeled und 15N-labeled peptides between the two scans. On the left it
almost looks like a 1:1 ratio, whereas on the right the 15N-labeled peptide
looks more abundant than the non-labeled peptide.
>> All peptide isotopolgues are ionized at the same time and have an identical
matrix since they enter the mass analyzer at the same time.
>> Can someone explain the obvious differences in isotopologue signal
intensities between the two scans?
>>
>> Thanks much!
>>
>> Chris
>>
>> ― 1 file ―
>>
>> □ H4.pdf (316kb)
>> http://list.abrf.org/r/file/mBFjR2h3dy0XjErMRAhC2OPKYiu-1kfX-2PdeohZ/
>>
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> --
> Priv.-Doz. Mag. Dr. Goran Mitulović, Ph.D.
> ORCID 0000-0003-1964-3965
> Head Proteomics Core Facility
> Medical University of Vienna
> Clinical Department of Laboratory Medicine
> Proteomics Core Facility, E5, LS5H
> Waehringer Guertel 18-20
> A-1090 Wien, Austria
> Tel: +43 1 40400 53750
> Mail: <email obscured>
> Web: www.meduniwien.ac.at/proteomics-core
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--
Priv.-Doz. Mag. Dr. Goran Mitulović, Ph.D.
ORCID 0000-0003-1964-3965
Head Proteomics Core Facility
Medical University of Vienna
Clinical Department of Laboratory Medicine
Proteomics Core Facility, E5, LS5H
Waehringer Guertel 18-20
A-1090 Wien, Austria
Tel: +43 1 40400 53750
Mail: <email obscured>
Web: www.meduniwien.ac.at/proteomics-core
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