Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry: Protocol Standardization and Database Expansion for Rapid Identification of Clinically Important Molds

Abstract

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can be used for routine identification of clinically important molds.

MALDI‐TOF MS‐Based Identification of Melanized Fungi is Faster and Reliable After the Expansion of In‐House Database

“…In this study, Epicoccum nigrum , Lasiodiplodia theobromae , Medicopsis romeroi, Rhytidhysteron rufulum, Roussoella percutanea failed to sporulates. Although PCR based sequencing is the gold standard for the identification, sometimes pigmented DNA (melanin) inhibits the amplification and limited sequence database may reduce the efficiency of identification of melanized fungi . The PCR inhibition by pigmented DNA was also observed in this study.…”

“…The protein extraction protocol was followed for melanized fungi was similar with some modifications of our previously standardized protocol . Two growth condition (in solid and liquid medium) were compared and solid media method used for this study.…”

“…The suspension was centrifuged at 10 000 rpm for 5 min and 4–6 μL of the supernatant was spotted (1.5–2 μL each time for 3–4 times) on to the surface of a 96‐target polished steel plate and allowed to dry. The dry spot was overlaid with 1 μL matrix solution (50 μL ACN, 47.5 μL DDW, 2.5 μL trifluoroacetic acid, and 1 mg α‐cyano‐4‐hydroxycinnamic acid) and subjected to MALDI‐TOF MS analysis …”

“…After the check, at least 20 best spectra were imported into MALDI Biotyper software and processed (Adjust mass, smooth, subtract baseline, Normalize, pick peaks, and batch processing) prior to include into the database. Then the spectra of each isolate were saved into the Bruker Biotyper database as the main spectra profile (MSP) of newly included isolates …”

“…In the past few years, matrix‐assisted laser desorption/ionization–time‐of‐flight mass spectrometry (MALDI‐TOF MS) emerged as a rapid diagnostic technique in clinical microbiology . Multiple studies have reported an MALDI‐TOF MS‐based identification of bacteria, yeast, and hyaline fungi . However, only a few studies have reported a MALDI‐based identification of melanized fungi due to the lack of a standardized protein extraction protocol and a limited database availability .…”