I have had experience with the MALDI III, MALDI IV and MALDI 4-DE and
successfully ran BSA on all three instruments. The delayed extraction
option enhances the ability to run proteins remarkably. On the DE version
of MALDI 4, I have had good results with 80kDa and had encouraging results
at just over 100kDa; the 80kDa did not run on MALDI III (no DE). As ever,
sample preparation is important for real samples, but start by getting good
results for BSA before proceeding to unknowns. My treatment of standard
samples is as follows.
Matrix 10mg/ml sinapinic acid in 50% acetonitrile, 0.1%TFA. BSA stock
solution at 100micromolar in water. Mix a volume of the stock (and serial
dilutions of it) with an equal volume of matrix and apply 0.5ul to the
sample slide. Dry in air at ambient or in the Kratos dry box. Then wash the
sample with 2 x 4ul water, wicking off the water after apporox 20secs, with
a tissue. Dry again and apply 0.5ul of matrix on top of each sample. Dry
again and analyse.
Comments:
1. Premixing sample with matrix is preferable to mixing on-slide by a
pipette; results are more consistent.
2. The washing step is essential to get sharp peaks.
3. The reapplication of matrix boosts the signal.
4. Matrix should be made fresh. Stock BSA is OK for a few days (store cold)
but make the dilutions fresh. If BSA is to be used over a long period,
acidify to 0.1%TFA to prevent bug growth.
With BSA, I get good signals from 2pmol upwards (MALDI 4, DE), but you may
need more on a MALDI III if you do not have delayed extraction.
If BSA is difficult, move down the scale to aldolase (39,212Da); it works well.
For nucleic acids, our practical limit is 40 bases long (HPA matrix). A
poster on this subject using a MALDI III is on my web site (see below)
Good luck
Len
> Greetings there,
>
> our laboratory has bought a Kompact MALDI III machine
>three months ago, so we are still very greenhorn to MALDI. We are quite
>succesfull with low mass compounds and oligomers, but totally unsuccesfull
>with proteins and NAs - e.g. BSA, we have tried all possible matrices (from
>sinapinnic till TRIS), all to us known methods of sample preparations, but
>per ten sample spots we got only one reasonable result, and it seems to be
>not good: 67720 Da for BSA.
> Where is the mistake we did and do? Can someone help us, please?
>
> Jan Havlis,
> Dept. of Analyt. Chem.
> Fac. Sci. Masaryk University (URL http://www.sci.muni.cz/)
> Brno, Czech Republic
>
*********************************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Old Addenbrookes Site
Cambridge, CB2 1GA, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 766002
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html