Re: Strange polymer signals
Marcus Macht (macht@sg17.chemie.uni-konstanz.de)
Wed, 21 Oct 1998 13:33:42 -0600
> Dear ABRF's
> I'm mainly involved in identification of 2D separated proteins.
> The procedure for the identification that is used is in general the
> following :
> 1. protein spot of intrest is digested in gel by trypsin
> 2. peptides are extracted
> 3. PorosR2 clean-up/concentration in a eppendorf Geloader tip
> 4. NanoSpray-ESI-Q-TOF- MS and MSMS, using micromass gold coated
> needles
>
> During NanoSpray-ESI-Q-TOF- MS the most important experiment is the MS
> scan. Parent ion scanning can not be used (for the moment) on the Q-TOF
> mass spec so that I'm totally dependent on the signal to noise in the
> primary scan.
> At this point a problem arise namely the peptide related multiple charged
> ions are obscured by polymer like peaks especially in the region below
> 700m/z. These polymer peaks are probably extracted (spray solvent =
> 60%MeOH/1%HCOOH) from eppendorf tubes because working in glass tubes
> gives a much cleaner spectrum. After a while (a few hours) even doubly
> charged polymer peaks are observed in the MS spectrum
>
> Here an overview of the most intense m/z values that are observed in the MS
> scan :
> serie 1 : 307.71 _ 409.17
> serie 2 : 387.19_489.22_591.27_693.32_795.38
> after 5 hours in eppendorf tube
> serie 3 : singly charged series + doubly charge serie :
> 396.14_446.14_497.15_548.18_...
> serie 4 : ........
> serie 5 : ........
> all these series differ from each other by 22 da!
>
> accurate mass measurement of the polymer building block reveals a mass of
> 102.0513da!!
>
> Has anyone observed the same problem or a similar problem?
Dear Jos,
we have observed similar signals here in Konstanz in the MALDI spectra of
in-gel-digested proteins. Often we get a set of for signals between 600 and
1100 Da with a mass difference of 113 Da between each peak. Currently we have
several different theories for its origin:
a) these peaks come from the acetonitrile, is accumulated on the HPLC column
and comes off the column at high acetonitrile contents because we observe it in
late HPLC fractions
b) it comes from the eppendorf cups and is eluted due to a higher solubility in
high acetonitrile concentration solutions.
c) clusters of Iron and trifluoroacetic acid (maybe originating from HPLC)
I doubt if our signals are identical with yours, but maybe they are of the same
or similar origin and the differences are caused by the use of, for examples,
different separation media (e.g. silicones) in the production of the plastic
cups. If you'll find an definite answer to this question, it would probably be
a point of general interest because I assume these (or similar) signal are
observed quite often.
Sincerely yours,
Marcus macht
--
* Marcus Macht, AG Przybylski, Faculty of Chemistry, University of Konstanz
* Box M 732, 78457 Konstanz, Germany
* Tel:++49-7531-883391 / Fax:++49-7531-883097
* Email: macht@sgiclu.chemie.uni-konstanz.de
* URL: http://www.ag-przybylski.chemie.uni-konstanz.de/~macht