Hello Thomas,

you are talking about reduction of steps and sample dilution. Do you wish to perform 2 Step digestion with LysC/Trypsin without any reduction alkylation before or after digestion? By omiting the reduction/alkylation step you will probably reduce the peptide recovery and the MS identification rate.

Are you digesting your sample in a denaturing condition i.e. with an urea or urea/thiourea buffer? then it will be necessary to make a dilution step to activate Trypsin activity. It doesn´t makes sense to omit this step.

Best, Murat

Hi Thomas,

it seems useless to do reduction/alkylation after digestion, unless you would like to test your sample in two situations: digestion without any reduction/alkylation (before or after digestion) and digestion after reduction/alkylation step.

Thanks Murat and Newton,

A bit more background :-) Currently, the sample is denaturated in 8M Urea , before dilution,reduction (DTT) and alkylation (IAA), and then digestion with trypsin (usual protocol). Eventhough we get incomplete digestion, we also want to shorten the digestion time so we thought to use LysC-Trypsin instead of trypsin only. Recommended conditions for LysC is 6M Urea so it would be possible to initiate digestion immediately after denaturation then dilute to de-inactivate trypsin (overall digestion of 4h instead of O/N). Peptides reduction-akylation is still necessary to break remaining C-C and to reduce sample complexity (ie make sure all Cys are in the same state).

I do not see why it would not work and I am pretty sure I have seen this being done but can't find any pointer. Anyone who convinces me that the idea is stupid will be rewarded :-)

Dear Thomas

2 Step Digestion with Lys-C and Trypsin is a common technique in LC-MS based proteomics. But it doesn't make sense to do reduction/ alkylation after digestion, since, as you have also mentioned it is necessary to linearize the proteins by opening the disulfid bonds at cysteins so that the enzymes will have access to K or R AAs which were protected from digestion because of the secondary structure due to the cysteine disulfid bonds. We have performed an experiment were we could improve peptide coverage from digested Antibodies to 86% by using reduction/ alkylation compared to 38% in unreduced samples.

In the 2 Step digestion protocol that i am using very frequently I reduce my samples for 30 min at RT with DTT, followed by alkylation with IAA for 20min at RT and samples were kept in dark. Than the undiluted sames were predigested with Lys-C for 3h at RT, diluted 4 fold with ABC buffer ( this step brings the sample condition to a trypsin active urea concentration) and digestion over night at RT.

Thanks Murat, Makes complete sense to open the IIary fold before digestion. Three more questions then :-). Concentration for DTT and IAA (and you standard protein concentration) and do you quench excess IAA with DTT before moving to LysC digestion? Cheers

Hello Thomas,

the amount of my protein starting material is approx. 10µg, but this protocol has also worked fine for me with a total protein amount of 50µg.

I have stock solutions of DTT and IAA of 0,5M and add 1µl of this to a volume of up to 100µl which gives a final concentration for DTT or IAA of about 5mM each (note that my IAA concentration is about 3 times less than given in other protocolls but I never have issues with this)

I never used an additional DTT Step for quenching the excess IAA (which is lower as mentioned above).

And finally, i would recommend a desalting procedure before going into the LC-MS (i.e. stage tipping)

Best, Murat