Generally trypsin is used for digestion of proteins before analysing them by MALDI-TOF MS. But if a protein or peptide, which inhibits trypsin is to be anlysed, then how the experiments are performed. Are there any other techniques available?
It hard to do Top-down by using MALDI-TIF. Just try different enzyme. There are several sites-specific proteolytic enzymes available now. Usually, trypsin is the most popular or the best one. But other still work.
Hi Ekant
You can still do bottom-up proteomics using acid digestion (with formic acid, TFA or HCl)
There are many protocols to do microwave assisted acid digestion of proteins, using a commercial or household microwave.
Only drawback is that you need a search engine that allows no enzyme in your peptide mass fingerprint analysis
Hello Ekant,
Since you cannot use trypsin, there are indeed other proteases available, or chemical digestion (non-specific as described by leonardo, or specific using cyanogen bromide, cleaving at c- extremity of methionins).
On the other hand, MALDI-TOF top-down sequencing is also a very interesting method
Basically, you use a MALDI matrix, forming radicals upon desorption, that cause an in source decay of the protein, giving a single cleavage of the protein chain. That allows you to get fragments of statistically all lengths of amino acids, and thus allows you to count the mass from one fragment to another and thus sequence the protein with resolution on the level of the residue (reads of up to 80 amino acids in total, with roughly 20-50 amino acids from each end of the protein)
(a paper about which matrix to use and other practical information)
good luck, hope it helps
http://pubs.acs.org/doi/abs/10.1021/ac070849z
it depends on the nature of inhibition(reversible?) if it is competitive you can compensate it by using higher substrate concentration or less enzyme
if it is not competitive, actually sufficient proteins should be digested for your further MS experiments
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