I'm doing my doctoral research and I'm working with mycobacteria, both tuberculous and non-tuberculous. One of the aims is to improve the identification of these microorganisms quickly and accurately. For this we use the MALDI-TOF MS (Bruker) analysis methodology. We were getting good results with an on-site protocol, but in the last few attempts I haven't been able to get good scores for my samples. In an attempt to identify the problem, I repeated previous samples that had given good results, changed reagents (HCCA matrix, formic acid, etc.) and reviewed all the steps in the process. To my surprise, I couldn't get the same good results as before, I repeated all the samples with fresh cultures and I still couldn't identify the problem. I would like to know if anyone else is having problems identifying or analyzing proteins with MALDI-TOF? If so, how are you coping? Any tips on how to get around this?

The equipment I use is a microFlex, the libraries were updated in 2023. I do the analysis in a 96-well plate, each sample goes in quintuplicate.

I have tested samples such as Mycobacterium bovis, M. tuberculosis, Mabscessus, M. chelonae, M. bolleti, M. avium, M. fortuitum, M. kansasii, among others. Only M. fortuitum has returned good results (>2,000). Most of the others did not return protein peaks (no peaks found).