Identification of Histoplasma-Specific Peptides in Human Urine

Histoplasmosis is a severe dimorphic fungus infection, which is often difficult to diagnose due to similarity in symptoms to other diseases and lack of specific diagnostic tests. Urine samples from histoplasma-antigen-positive patients and appropriate controls were prepared using various sample preparation strategies including immunoenrichment, ultrafiltration, high-abundant protein depletion, deglycosylation, reverse-phase fractions, and digest using various enzymes. Samples were then analyzed by nanospray tandem mass spectrometry. Accurate mass TOF scans underwent molecular feature extraction and statistical analysis for unique disease makers, and acquired MS/MS data were searched against known human and histoplasma proteins. In human urine, some 52 peptides from 37 Histoplasma proteins were identified with high confidence. This is the first report of identification of a large number of Histoplasma-specific peptides from immunoassay-positive patient samples using tandem mass spectrometry and bioinformatics techniques. These findings may lead to novel diagnostic markers for histoplasmosis in human urine. 1. Introduction Infection and disease caused by pathogenic and opportunistic fungi has been increasing over the past decade. One widespread and severe fungal disease, histoplasmosis, is caused by the dimorphic fungus Histoplasma capsulatum. The H. capsulatum mycelial phase may be inhaled by mammals to cause pulmonary disease [1]. Disease progression then occurs as the fungus transforms to yeast phase and disseminates to other parts of the body [2]. While most infections are mild, it can lead to rapid progression and life threatening symptoms for individuals with weak immune systems such as elderly, infants, HIV, and cancer patients or transplant recipients [3]. Furthermore, once infected, a latent infection may also be reactivated. Current laboratory testing for histoplasmosis does not provide adequate clinical sensitivity and specificity for accurate diagnosis of this infection. Antigen cross reactivity with other fungi species can be especially troublesome [4, 5]. Although progress has been made in understanding the pathogen-host interaction [6, 7], there are currently no known biomarkers of histoplasma infection, whereby molecular or chemical structures have been clearly defined. Incomplete genomes and/or databases for either protein or nonprotein compounds from Histoplasma have been a significant limitation of the work to date. This limitation has been improved due to recent efforts of the Human Microbiome Project hosted by the Genome Center at