Hi Rampai,

 I think it  depends on what you want to characterized in the Ascomycota group. Do you want to identified protein or metabolite profiles?  Would it be for identification and clustering inside the group? or do you want to detect  production o determined molecules?

Maybe this paper is useful for you work. 

Good luck,

Diana

Article MALDI-TOF mass spectrometry identification of filamentous fu...

Hello Rampai,

Unfortunatelly, Identification of fungi by MALDI-TOF doesn't work as well as for bacteria. One way is to use Sabouraud liquid culture medium under gentle stirring to avoid sporulation. If the signal is too low, an extraction is needed (ethanol/TFA/Acetonitrile).

Good luck,

Romain

Hi all,

Thank you very much for all answers. Since I am quite new to this method so I just would like to get more information before I start my experiment. My purpose to do this is to provide the spectra of my fungal strains to the database and make it available for fungal identification in the future since there is very few reference spectra of Ascomycota and their anamophs in the library/database right now. However as there are many factors influencing the protein profile i.e. age of culture, fungal parts, treatment conditions and extraction method so I just would like to know and make sure about the optimum condition that most people have been done before for my information.

Thank you very much once again.

Regards,

Rampai

I am not sure if MALDI-TOF will work for fungal identification.

Please refer to this paper on identification based on DNA sequencing.

    http://jhs.pharm.or.jp/data/49(6)/49_531.pdf

Hi Rampai,

Yes, the age of the culture will affect the spectral profile, though in most cases, the peaks that are specific for the species are not affected much. The most intense peaks between 2 to 20 kDa correspond with the ribosomal proteins and these are expressed regardless of the passage of the culture, though you could see different levels of expression. This means that if you are only interested in species identification, the number of passages will have a minimal effect on the results. Your identification will be a bit more reliable if you include both high and low passage Ascomycota in your reference spectra as this allows you to identify the proteins that are expressed regardless of passage.

 However, if you are strain typing, you will also need the smaller and less intense peaks and the variability between strains usually occurs in these proteins. These are affected very much with the number of passages. If you would compare a group of high passage strains and low passage strains, they will more or less cluster according to passage (see paper in the link) . The expression pattern of a lower passage strain is in general closer to the in vivo situation. This usually means more proteins are expressed, so you have a higher chance of differentiating strains with low passages.

Article Comparison of MALDI-TOF mass spectra with microsatellite len...

This depends on the organism. It is important to get the fungus developing enough biomass that's why it will vary between different groups. Slow-growing fungi will need more time, e.g. it generally takes longer for dermatophytes.

Identification with this technique works good as soon as you can elaborate proteins from the cells. There is also data suggesting that the majority of routinely detected proteins are ribosomal proteins. So, if you need to detect a different protein you may need to adjust cultivation conditions to better see it in the resulting profile.