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Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris

ABSTRACT: Cordycepin, which has great immunomodulatory activities such as anticancer, antifungal, antivirus, antileukemia and lipid-lowering ones, is the secondary metabolite of Cordyceps militaris (C. militaris). Liquid submerged fermentation is the common cultivation process to produce cordycepin. To optimize the fermentation process and improve production, monitoring the cordycepin secretion in the fermentation is essential. The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation, so more rapid and convenient methods are required. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is more attractive for faster and direct detection. Therefore, MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium. This method made accurate quantification of cordycepin in the range of 5–400 μg/mL with a relative standard deviation of 5.6%. The recovery rates of fermentation samples after the 1, 13, and 25 days were 90.15%, 94.27%, and 95.06%, respectively. The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20th culture day, respectively. The cordycepin secretion curve of the liquid fermentation of C. militaris was real-time traced over 25 days. Graphical abstract Image 1 Highlights • A rapid quantification method of cordycepin based on MALDI-MS is proposed.• The quantification relies on the stable isotope standard method.• Rapid determination of the cordycepin content in the liquid fermentation broth of Cordyceps militaris without pre-treatment.• Monitoring the fermentation state of C. militaris fermentation broth is benefit to improve the yield of cordycepin.

SUBMITTER: Chen J

PROVIDER: S-EPMC8424359 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

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Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris

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