May 9, 2005 at 12:33 pm #861BobParticipant
I was hoping that someone on here will be able to give me a few pointers. 🙄
I thought that I would have a go at imaging using our MALDI instrument but I am having all sorts of problems – I just don’t see very much signal from my tissue samples no matter how I deposit the matrix. I have tried spraying directly onto the sample using a nebulizer as well as normal dried droplet but I never see anywhere near as many signals as I see in the literature. What could be going wrong?
I have tried various concentrations of both Sinapinic and Alpha, blotting onto a (metal) target plate, washing the sample in ethanol etc but the most I ever see is maybe 5-10 proteins at any one time (with only a couple of ‘strong’ peaks). The tissue sample is rat brain by the way. The one thing that I thought that it might be is the thickness of the sample. I am using pretty thick samples (200-300um) as this is the best I can do with my existing equipment – could this be causing me problems (other than the obvious effect on resolution/flatness etc)?
Any help or pointers would be greatly appreciated! Or is this normal and the spectra in the literature are ‘best ever’ data – am I expecting too much?? 😕
BobMay 11, 2005 at 12:36 am #863chaurapParticipant
Your problem is effectively the thickness of your sample. Early work that described the analysis of microdissected tissues such as neurons only yielded very few lower MS signals when 2-5 DHB was used as matrix. This is in parts due to the presence in the tissue of high salt contents affecting local matrix crystallization. A thickness of 300 um is extremely thick with respect to the ~10 um that are routinely cut from fresh frozen tissue blocks. In most cases, analysis of 10 um thin sections with sinapinic acid as matrix yield high quality data in the MW range from 2 to 30 kDa with some signals detected in some cases above 100 kDa. When using SA as matrix in combination with a TOF MS equipped with a 337 nm nitrogen laser, from a single tissue coordinate, one should see anywhere from 200 to 500 m/z signals. I strongly recommend that you get in touch with your local pathology lab. I guaranty that they have a cryostat, which should allow you to cut thin sections from your samples.
PierreMay 13, 2005 at 2:53 pm #867BobParticipant
Thanks a lot for your advice Pierre. 😀
I have been discussing the matter with the guys who performed the sectioning for me. It looks like they had been sectioning the sample at room temp rather than cryogenically freezing the sample first. Would I be right in thinking that you will lyse more cells (and hence obtain more protein/peptides on the tissue surface for imaging) if the section is performed on a frozen sample and could this perhaps be the cause of my problems rather than the thickness? Or should the temperature not make much difference?
Any further help would be much appreciated! 😉
BobMay 13, 2005 at 7:14 pm #868chaurapParticipant
Most of the tissue profiling and imaging work has been performed on thin sections cut from fresh frozen samples. I recommend reading the two references below for protocols.
Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation. Schwartz SA et al, J. Mass Spectrom. (2003) 38: 699-708.
Integrating histology and imaging mass spectrometry. Anal. Chem. Chaurand P. et Al, (2004) 76(4): 1145-1155.
You can also work with fresh (non-frozen) tissues. In this case, one approach is to blot the protein on hydrophobic surfaces. Please refer to:
Direct Profiling of Proteins in Biological Tissue Sections by MALDI Mass Spectrometry. Chaurand P. et Al, (1999) 71(23): 5263-5270.
If you need the pdf’s, I can send them to you.
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