Elsevier

Analytical Biochemistry

Volume 411, Issue 1, 1 April 2011, Pages 122-128
Analytical Biochemistry

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

https://doi.org/10.1016/j.ab.2010.11.025Get rights and content

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI–TOF and MALDI–qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250 ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H]+ ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI–MS; however, these fungi may be successfully analyzed by MALDI–TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI–TOF MS.

Section snippets

Reagents

Acetonitrile (high-performance liquid chromatography [HPLC] grade), sodium citrate, and hydrochloric acid (6 N) were acquired from Fisher Scientific (Fairlawn, NJ, USA). Tricyclazole was acquired from Wako Pure Chemical Industries (Osaka, Japan). Malt extract agar (MEA) was acquired from Difco (Sparks, MD, USA). Deionized water (DDI) was produced with a Synthesis A-10 system (Millipore, Billerica, MA, USA). All other reagents were acquired from Sigma–Aldrich (St. Louis, MO, USA), and all

Results and discussion

Darkly pigmented fungi, such as A. niger, yield poor fingerprint mass spectra that contain only a few peaks of low relative abundance (Fig. 1). It is hypothesized that the presence of photo- and chemoprotective pigments, such as melanin, in the cell wall may suppress ionization and ultimately the MALDI-based detection of expressed proteins in the sample. To test this hypothesis, we examined the effect of spiking synthetic melanin into standard protein and peptide solutions. We observed

Conclusions

The presence of fungal melanin in MALDI sample deposits suppressed peptide and protein ion signals during the acquisition of MALDI–TOF mass spectra. Suppression of standard peptide and protein signals was observed on the addition of both commercially available synthetic melanin and melanins purified from A. niger to the MALDI sample. Suppression of peptide and protein [M+H]+ ion signals was observed at melanin concentrations greater than 1250 ng/spot, a concentration significantly less than the

Acknowledgments

The authors thank Diane Schwegler-Berry for help with the preparation and analysis with FESEM. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health. This work was supported in part by an interagency agreement with the National Institute of Environmental Health Sciences (Y1-ES0001-06).

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