RE: mass spec: maldi of phage coat protein

Amina S. Woods (awoods@jhmi.edu)
Sun, 28 Nov 1999 12:25:32 -0500

On Friday, November 26, 1999 4:44 AM, Matthew Sampson
[SMTP:mps36@mole.bio.cam.ac.uk] wrote:
> Dear ABRFrs:
>
> I'm hoping someone might have some ideas on how to prepare a sample for
> maldi in the following situation:
>
> I am working with intact phage and get strong signal for the coat protein
> (46 AA's, 5kDa) in aCN and a variety of other matrices. After performing
a
> chemical cleavage at AA37, a 1000 dalton C-terminal fragment is generated
> and both the 5 kda parent and the 1 kda are visible in the spectra.
> Allowing the cleavage to continue to excess results in almost complete
> depletion of the parent in the maldi spectra which are dominated by a
strong
> peak from the 1kda fragment.
>
> The problem is that I have never seen the other fragement (4 kda) that
would
> be generated in the cleavage. The abundant presence of the small
fragment
> indicates that the large part must also be there, but it never seems to
fly.
>
> The small fragement (9 residues) contains both K and R.
>
> The large fragment contains mostly G,A,V,L,I with a few D,F,N,Q,P,S,T.
> There are no K, R or H.
>
> If any one has any tips on how I might get signal out of this larger
> fragment, they would be greatly appreciated.
>
> Cheers,
>
> Matthew
>
>
>
>
>
>
> Mr. Matthew P. Sampson
> Department of Biochemistry
> University of Cambridge
> Tennis Court Road
> Old Addenbrookes Site
> Cambridge CB2 1GA UK
> Tel: 01223-333638/766039
> Fax: 01223-766002
> International: +44-1223-333638/766039 (fax:-766002)
>
> http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
> http://www.bio.cam.ac.uk/dept/biochem
Matthew:Prepare your sample the following way:

0.3ul sample+0.3ul 25mM Ammonium Citrate pH 6.5-7.0+ 0.3ul
alpha-cyano-4-hydroxy-cinnamic acid (saturated solution in 50%ethanol).

If this does not work replace the amm citrate with the supernatant of a
saturated solution of ammonium sulfate. Let us know if any of the advice
you receive works.

Amina

Amina S. Woods Ph.D.
Johns Hopkins School of Medicine
725 N Wolfe St. Baltimore, MD 21205
Tel: (410) 614-4981, Fax: (410) 955-3420
E-mail: awoods@jhmi.edu