I have not used sinapinic acid but matrix that consists of α-cyano-4-hydroxy-cinnamic acid (10 mg/mL solution in 50% CH3CN, 0.1% TFA in H2O). The samples were diluted in 1:1 ratio in the matrix when applied on the MALDI-TOF MS sample plate e.g. first pipet the sample matrix on the plate and then the sample in the same drop (with some speed before the matrix dries). This is for shorter peptides with MW under 10 000. Sinapinic acid suits for bigger proteins but then from what I read you should use 70% acetonitrile instead. The sample will dry and leave precipitate. In my lab, the sample if further dried with blowing a little bit air with a heat gun on the plate. Just make sure that your matrix solution is rather fresh to get the best possible signal.

Hi Gaurav,

The precipitate you are observing is due to the presence of Tris and NaCl.  Even if the MALDI-TOF MS is tolerant towards additives, the presence of salts will give rise to "caking" of the co-crystallized sample... 

If the protein is under ~20kDa, you can try and de-salt it with a C4 containing clean up material eg., Empore disk (by 3M) or Zip-Tip (Millipore).  Elute the protein/polypeptide in ~40% Acetonitrile/0.1% TFA in a small volume (eg., 5 microliter) and this material should form uniform co-crystals with the matrix.

Good luck, 

Hediye

Thanks Susanna and Hediye for the inputs. 

My proteins size is approx 28 kDa and 26kDa. I have observed two things during the analysis. The precipitated samples have more noise near the desired molecular weight region although smaller peaks but non precipitated sample gave clean peak with one or two minor peaks far from the desired molecular weight region(very small molecular weight due to degradation during ionisation I guess).

My question is, does precipitation leads to noise? or extra peaks near the desired peaks? 

Is there a difference between the samples - why do you think some of the samples precipitate and some don't? You can try to mix the sample while pipetting on the matrix solution. Also, you can toggle the laser intensity slightly to see if you can get a better peak.  

Hi Gaurav,

With MALDI-TOF, when analyzing proteins, it is usual to see doubly charged species at m/z corresponding to half the predicted molecular weight.  It is also usual to see triply and quadruply charged species.  So, have an open mind when interpreting the spectum.  This happens espeically when your laser attenuation is high.  

Best,

Hediye

Thanks Hediye for the clarification. High laser attenuation may lead to variable charge distribution which may further lead to background noise or multiple peaks near the desired region.

@Susanna- all the samples are proteins with differing isolectric points. Thanks for your suggestion.