Methodological Determinants of Fungal Identification by Proteomic Mass Spectrometry: A Narrative Review with Emphasis on MALDI-TOF MS

Abstract

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become the dominant proteomic platform for fungal identification, turning a multi-day, morphology-dependent workflow into a minutes-long protein-fingerprinting assay. Yet reported performance for the same genera ranges from under 20% to over 95% depending on the study. This narrative review examines sources of heterogeneity across five domains: (1) biological and pre-analytical properties, including colony age, growth phase and dimorphism, culture medium, incubation temperature, pigmentation, and taxonomic complexity; (2) sample-processing variables, including protein-extraction chemistry, deposition technique, and matrix selection; (3) instrument-acquisition parameters, including laser intensity, shot number, signal-intensity thresholds, calibration, and cross-platform differences; (4) database and bioinformatic factors, including reference-library completeness, score-threshold interpretation, and software version; and (5) specimen type and clinical context, particularly for culture-independent workflows. The review examines how these factors affect proteomic identification of molds. First, variability attributed to fungal biology may be confounded by methodological variability: studies using standardized, validated extraction protocols may report less variability attributable to biology than studies using less-controlled methods. Second, reference-library completeness and standardized, locally validated sample preparation and deposition are particularly actionable determinants. Third, an unresolved challenge is reliable identification of rare, cryptic, melanized, dimorphic, and emerging organisms. The review concludes with evidence-informed quality-assurance practices considered in the context of CLSI M58.