Improved sample preparation for intact cell mass spectrometry (biotyping) of mycelium samples taken from a batch fermentation process of Penicillium chrysogenum.

Rationale: Penicillium chrysogenum is an important species in biotechnology and an improved production rate for penicillin drug variants is of utmost interest. Intact cell mass spectrometry (ICMS) or biotyping can be a novel and time-saving tool to monitor a fermentation process of Penicillium strains for fast intervention during penicillin production.

Methods: Fermentation broth was collected directly from a fermenter at specific time points known to show significantly different penicillin production rates. The mycelium was purified by washing multiple times with water and recovered by centrifugation. The mycelium was further mixed with matrix-assisted laser desorption/ionization (MALDI) MS matrix and immediately spotted on different types of targets. ICMS spectra were obtained by MALDI time-of-flight (TOF) MS in the positive ion linear mode in the m/z range 3000 to 16 000.

Results: An ICMS method for culture broth samples of P. chrysogenum was developed. It was shown that ferulic acid mixed with sinapinic acid (2.5 mg and 22.5 mg/mL) is the most appropriate matrix combination. The matrices were dissolved in acetonitrile/0.1% trifluoroacetic acid (70/30, v/v) and spotted together with the sample on various target types. Sample preparation was thoroughly studied for homogeneity and reproducibility.

Conclusions: Culture broth directly collected from a bioreactor could be analyzed applying the optimized approach. The ideal choice of matrix, the adequate preparation technique and the type of target were the focus of this work showing that samples collected at different times during fermentation exhibit a characteristic pattern using the developed method.