I am working with proteins and glycoproteins with a MALDI-TOF instrument for the determination of intact mass.
For proteins, I work with SA, sDHB or HCCA depending on the molecular size.
Currently I am working with big glycoproteins (200 kDa around).
I tested with SA but the signal is not so nice.
Is there another matrix or mix of matrices for glycoproteins.
MALDI:UltrafleXtreme Bruker
Best,
Sébastien
Hi Sébastien
both SA and sDHB should work, which concentrations are you using,
you should consider using an AncorChip target plate to concentrate your sample, further you should tune your MS device for high masses. Nevertheless, 200 kDa is challanging, and the main question is what you intend to see?
best
Bogdan
Hello Bogdan,
Basicallly we have developped on my core facility a procedure to assess the quality on a protein using dynamic light scattering, UV spectrum, MALDI-TOF (kind of turnkey service).
For this sample I am working with 2.5 mgml, concentration that the user gives me. Sample was eluted in SA on a C4 Ziptip.
The goal here is to improve the peak signal to get a better precision on the mass. After that, we would like to calculate "briefly" how many sugars are linked to the protein.
Thank you for your help,
Best,
Sébastien
hi Sebastien
I can image, you should try to prepare using a SA thin layer, see my ABC paper, you need to optimize your lens voltages and delay times 600 ns on your ultraflex
best
Hello Sébastien,
you may try THAP (2,4,6-trihydroxyacetophenone) which is good for glycoproteins. You should dissolve 7,5 mg in 1 mL of ACN with 20 mM DAC (Diammonium hydrogen citrate).
However, even with optimal sample preparation and equipment tuning, linear mode resolution will be an important limitation to you achieve what you need (number of sugars). At that mass range you won´t see acurate peaks to support a glycosilation profile of your protein.
Since your sample seems to be very nice, regarding concentration and purity, I would plan a digestion with glycosidases and then look for glycans in linear/reflectron modes.
Thank you for your answers. I gonna try to investigate these options. Of course the discussion is still opened to other possibilities.
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