Rapid glycosyl-inositol-phospho-ceramide fingerprint from filamentous fungal pathogens using the MALDI Biotyper Sirius system.

RATIONALE: Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are known to be major lipids in plant and fungal plasma membranes and to play an important role in stress adaption. However, their analysis remains challenging due to the several steps involved for their extractions and purifications prior to mass spectrometric analysis. To address this challenge, we developed a rapid and sensitive method to identify GIPCs from the four common fungal plant pathogens Botrytis cinerea, Fusarium graminearium, Neurospora crassa and Ustilago maydis. METHODS: Fungal plant pathogens were cultured, harvested, heat-inactivated and washed three times with double-distilled water. Intact fungi were deposited on a matrix-assisted laser desorption ionization (MALDI) target plate, mixed with the matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid solubilized at 10 mg/mL in chloroform-methanol (9:1 v/v) and analyzed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were acquired from m/z 700 to 2000. RESULTS: MALDI time-of-flight (TOF) mass spectrometric analysis of cultured fungi showed clear signature of GIPCs in B. cinerea, F. graminearium, N. crassa and U. maydis. CONCLUSIONS: We have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with an apolar solvent system to solubilize the matrix is applicable to the detection of filamentous fungal GIPCs.

The control of translational accuracy is a determinant of healthy ageing in yeast.

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.