Dear All,
This is a very peculiar peptide, loaded with Gly and Cys (18 of 38 res.).
The aa composition does not leave much for analysis.
Reliable values should be obtained for Asx (2 equivalents), Glx (2 eq), Pro
(3), Ala (1), Leu (1) and Lys (1 by MS or 2 on paper!).
Ser will be (should be) low; Gly is almost never reliable!; Cys is Cys -
even modified-cys is tricky; Tyr often low due to oxidation/acid hydrolysis;
Trp is destroyed. -- Sometimes Pro is Questionable.
If the comp data clearly demonstrates equivalent amounts of Asx, Glx and Lys
and the correct ratios of the other reliable values then there is a problem.
If so, I would even speculate that because of the Gly (and Cys) content acid
hydrolysis may not be reliable. Have you done a time course for the aa
analysis.
Best regards,
alex
Alexander W. Bell
Anat. and Cell Biol.
McGill Univ
Montreal Quebec Canada
phone (514) 398-1393
----- Original Message -----
From: "Ioannis A. Papayannopoulos" <ioannis_papayannopoulos@mass-spec.net>
To: "Recipients of ABRF List" <abrf@aecom.yu.edu>
Sent: Saturday, December 09, 2000 9:14 AM
Subject: Re: MS & ProtSeq
> The measured molecular masses do correspond to a peptide with the
> expected sequence minus the C-terminal Lys and four disulfide
> bonds(calculated monoisotpic molecular mass 3680.24 and average mass
> 3683.06) - I am assumming that the mass measured by ESI with a Q-Tof
> instrument (3680) is monositopic whereas the mass measured by MALDI
> (3684) is average. I would not put too much emphasis on the AAA data
> and as for the Edman data, do you actually see Lys in the last cycle?
> Otherwise, I can not imagine how the C-terminal Lys might "fall off"
> during ESI and MALDI mass spec analysis.
>
> Regarding the number of charges in ESI, given the size of this peptide
> it is not unreasonable to expect to see a triply charged ion even
> without the third basic site (C-terminal Lys).
>
> As for the MS/MS data, the ion at m/z 204.14 can, indeed, be assigned
> as the y2 ion for the full-length sequence (WSG...YGK) but it can also
> be assigned as the GY or YG internal fragment ion minus ammonia. These
> ions, at m/z 204 (204.0661 calculated monoisotopic mass and 204.2
> calculated average mass, to be exact), would have nominal structures,
> respectively, [CH-CO-NH-CH-(CH2-phenol)-CO]+ and
> [CH=CH-(phenol)-CO-NH-CH2-CO]+, and given that there are several GY and
> YG pairs in the sequence, one would expect this ion to be quite
> abundant in the MS/MS spectrum.
>
> Ioannis Papayannopoulos
> AstraZeneca R&D Boston
> Worcester, MA
>
> On Thu Dec 7 2000 hi-Sheng LI (chembiology00@hotmail.com) wrote:
>
> >Dear Friends:
> >
> >
> >By Edman chemistry, One polypeptide with 38 AA residues follows the
> >sequence
> >as:
> >WSGCSPCPGNECCSKYGYCGLGGDYCGAGCQSGPCYGK
> >It fits quite well with the AAA results. The problem is:
> >1) Maldi MS showed the molecular weight 3684, in the case of ESI Q-tof
> >it's
> >3680, that's ok.
> >2) If we calculate the mw from the sequence above mentioned, we'll get
>
> >3811 (4 disuphide bridges assumed)
> >So, what's going on with the 131 Da part (3811-3680) during the MS?
> >
> >
> >If the C-terminal K was lost during ionization (? it seems not
> >reasonable?
> >any idea?), probably, the ion with mw 3683 may be observed. But
> three->charge
> >ion (1227.5) for 3680 was visualized in ESI MS, if K was lost, it
> >should not
> >be able to get three charges, am I right? And also MS/MS on the native
>
> >peptide (1840.7++) gave reasonable y2 ion 204.14.
> >
> >Any comments are appreciated!
> >
> >
> >
> >Shi-Sheng Li, Ph. D.
> >Biomedical Center, Box 579
> >Uppsala University
> >751 23 Uppsala
> >Sweden
> >Emails: chembiology00@hotmail.com; rockylee86@yahoo.com
>
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