Updated publication reference for PubMed record(s): 28580409, 26926564. Fungal proteomics is a developing field that requires renewed interest and attention from the scientific community in many aspects. One of the most compelling objectives of fungal biology is to find out how fungal pathogens colonize a host, in order to find new ways of impeding this colonization, and proteomics is an important and useful tool to this end. However, fungi are also taxonomically interesting and are important biochemical model organisms for the study of many cellular processes, such as cytoskeletal regulation. Additionally, fungi are sources of secondary metabolites, many of which serve as medications for humans. Nevertheless, much work remains in developing systems-biology approaches to understanding fungal gene expression and secondary metabolism. Current fungal proteomics approaches involve 2D SDS-PAGE and extensive, complex, protein extraction methodologies. In this work, an application of a modified Folch extraction to protein extraction was used to perform de novo peptide sequencing of the proteome from the plant and human pathogen Lasiodiplodia theobromae, which greatly streamlined and simplified the analysis process. Using a metaproteomics bioinformatics approach, many novel proteins for L. theobromae were identified and targeted for further biochemical characterization and annotation efforts.