Proteome of Penicillium funiculosum NCIM1228 and ∆Snf1 grown in glucose and Avicel in the presence as well as the absence of calcium

Penicillium funiculosum NCIM1228 and ∆Ampk were compared across calcium-limited and calcium-replete cellulosic conditions to assess changes within the proteome using quantitative mass spectrometry. These comparative proteomic data provide insights into cellular responses to produce and secrete cellulase during nutrient limitation, and the role of AMPK in cellulase secretion.

Methods
Profiling of the total proteome of NCM1228 and ∆Ampk was performed under calcium-limited and calcium-replete cellulosic conditions. NCIM1228 and ∆Ampk were cultured in Mandel’s medium having cellulose, or cellulose+calcium at 28˚C (120rpm) (in triplicates)(3). 10mL mycelial culture was harvested after 48h, and the remaining culture was incubated for another 72h to harvest the secretome. The secretomes were examined for the desired phenotype by SDS-PAGE and enzyme assays(4, 5). Once the distinct phenotypes associated with the two strains under given conditions were ascertained, the whole cell proteome was extracted from 48h mycelial samples as previously described in Randhawa et al (2023), and quantified by the BCA method. To assess the variance during sample preparation and measurements, we added 1µg 13C and 15N labeled human Apolipoprotein (Apo-1) (Sigma) to 50µg sample before processing. The samples were alkylated and reduced before proteolytic digestion with trypsin (1µg/50µg of protein) for 16h at 37˚C(6). Peptides were purified and concentrated by C18 spin columns (ThermoFisher Scientific). Purified samples were vacuum-dried and reconstituted in 0.1%(v/v) formic acid before proceeding for LC-MS/MS analysis using ThermoFisher Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer equipped with nano-LC Easy 1200.