Bacterial analysis by MALDI-TOF mass spectrometry: An inter-laboratory comparison

Abstract

Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed. has been used to analyze intact, cultured microorganisms with minimal sample handling. Two recent review articles, which include the capabilities and current limitations that need to be addressed, provide an excellent overview of this emerging research field [1,2]. The MALDI-TOF MS technique for identifying biomolecules provides rapid analysis time (Ͻ1 min per sample analysis), low sample-volume requirements (Ͻ1 L fluid), and the highly selective nature of mass-spectrometric analysis based on relative molecular masses. The m/z values for mass spectral peaks and the patterns with which they are observed provide very specific and unbiased analysis, as they indicate molecular weights of true components of the sample. Bacterial cells have been identified by comparing MALDI-TOF spectra obtained from cultured bacterial cells and simple microbial mixtures against a library of known MALDI-TOF spectral fingerprints obtained from intact bacterial cells [3,4] or from comparison with masses predicted from a proteomic database [5,6]. The proteomic approach has been demonstrated to correctly identify bacteria from spectra originating at different laboratories [5], however, this approach is currently suffering from an incomplete protein database for many of the organisms that are of interest. As the proteomic database becomes more populated with organisms of concern, this approach will become more feasible for bacterial identification, at

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

“…Complementary to the already determined intraspecies (4,36) and interlaboratory (37) 6.0 kV; detector gain, 1,650 V; and gating, maximum, 1,500 Da, for the ultraflex instrument. Second, the influence of different cultivation conditions was characterized by cultivation of the 10 strains on four different media (Columbia blood agar, chocolate agar, Mueller-Hinton agar, and tryptic soy agar; Heipha, Eppelheim, Germany) at 30°C under aerobic conditions for 48 h. Finally, the influence of the age of the bacterial cultures was investigated by analyzing three strains (B. aurantiaca DSM 4731, B. caribensis DSM 13236, P. aeruginosa DSM 50071) that were cultivated for 48 h at 30°C on Columbia blood agar and subsequently stored for 2, 5, and 7 days at room temperature.…”

Rapid Detection of Vancomycin-Intermediate Staphylococcus aureus by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…As 10 4 to 10 6 CFU of the test microorganism are generally required for analysis by MALDI-TOF MS (45,46), it is likely that the overwhelming majority of susceptible clones obscured the signal from the nonsusceptible ones. Although the hVISA phenotype is known to be unstable during freezing, we confirmed retention of the hVISA phenotype after freezing.…”

Comparison of Two Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Methods with Conventional Phenotypic Identification for Routine Identification of Bacteria to the Species Level

“…Because ribosomal proteins are part of the cellular translational machinery, they are present in all living cells. As a result, the MS protein fingerprints are not significantly influenced by variability in environmental or growth conditions (11) and encompass targets widely used for identification of bacteria to the species level (25).…”