Skip to Content
Merck
  • Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies.

Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies.

Applied and environmental microbiology (2014-05-06)
Maria-Theresia Gekenidis, Patrick Studer, Simone Wüthrich, René Brunisholz, David Drissner
ABSTRACT

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Formic acid, ACS reagent, ≥96%
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Sigma-Aldrich
Ethanol, purum, absolute ethanol, denaturated with 2% 2-butanone, A15 MEK1, ≥99.8% (based on denaturant-free substance)
Sigma-Aldrich
Ethanol, purum, fine spirit, denaturated with 2% 2-butanone, F25 MEK1, ~96% (based on denaturant-free substance)
Sigma-Aldrich
Trifluoroacetic acid, ReagentPlus®, 99%
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Sigma-Aldrich
Ethanol, tested according to Ph. Eur.
Sigma-Aldrich
Ethanol, purum, secunda spirit, denaturated with 2% 2-butanone, S15, ~96% (based on denaturant-free substance)
Sigma-Aldrich
Formic acid solution, BioUltra, 1.0 M in H2O
Supelco
Trifluoroacetic acid, analytical standard
Supelco
Acetonitrile, analytical standard
Sigma-Aldrich
Ethanol, for residue analysis
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Ethanol, BioUltra, for molecular biology, ≥99.8%, (absolute alcohol, without additive, A15 o1)
Sigma-Aldrich
Acetonitrile, electronic grade, 99.999% trace metals basis
Supelco
Ethanol, standard for GC
Sigma-Aldrich
Ethanol, purum, absolute ethanol, denaturated with 4.8% isopropanol, A15 IPA1, ≥99.8% (based on denaturant-free substance)
Sigma-Aldrich
Ethanol, ACS reagent, prima fine spirit, without additive, F15 o1
Sigma-Aldrich
Ethanol, purum, absolute ethanol, denaturated with 1% cyclohexane, A15 CYCLO1, ≥99.8% (based on denaturant-free substance)
Sigma-Aldrich
Ethanol, purum, fine spirit, denaturated with 4.8% methanol, F25 METHYL1, ~96% (based on denaturant-free substance)
Sigma-Aldrich
Formic acid, puriss., meets analytical specifications of DAC, FCC, 98.0-100%
Sigma-Aldrich
Trifluoroacetic acid, puriss. p.a., suitable for HPLC, ≥99.0% (GC)
Sigma-Aldrich
Formic acid, reagent grade, ≥95%
Sigma-Aldrich
Acetonitrile, biotech. grade, ≥99.93%
Sigma-Aldrich
Acetonitrile, ≥99.5% (GC)
Sigma-Aldrich
Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)
Sigma-Aldrich
Trifluoroacetic acid, ≥99%, for protein sequencing
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%