Mycobacterial inactivation protein extraction protocol for matrix-assisted laser desorption ionization time-of-flight characterization of clinical isolates

Morales, Manuel Pastor Forero; Lim, Chuan Kok; Shephard, Lisa; Weldhagen, Gerhard F.

doi:10.4103/ijmy.ijmy_104_18

Cited by 2 publications

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“…This may be explained in part by the limitations of the protein extraction procedure. The reasons for this are: (i) thickness and rigidity of the mycobacterial cell wall, which is made of a mycolyl–arabinogalactan–peptidoglycan complex structure with an outer layer of mycolic acids [ 4 ], which requires harsher methods for cell disruption and cytoplasmic protein availability; (ii) in the case of slow-growing mycobacteria, which have a lower metabolism and low number of ribosomes [ 12 ], the peak number and protein profiling are impacted; (iii) as a biosafety level 3 pathogen, mycobacteria such as M. tuberculosis have to undergo an inactivation step before protein extraction, which may also impact the methodological performance [ 10 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol

2023

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Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method’s efficacy for M. tuberculosis identification.

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Section: Introductionmentioning

confidence: 99%

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol

2023

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Systems for Identification of Bacteria and Fungi

Heaton,

Bhatti

2023

ClinMicroNow

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A proteomics‐based approach, matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS), was introduced in clinical microbiology laboratories between 2005 and 2010 as a highly reliable and much more rapid method for identifying bacteria and yeast. Advances in molecular tools have allowed proteomics and genomics to have a substantial role in the identification of bacteria and yeast in clinical laboratories. In many laboratories, MALDI‐TOF MS has become the primary method for bacteria and yeast identification, supplanting biochemical analysis, including automated methods. DNA target sequencing may be performed on organisms growing in pure culture. This is typically performed when automated systems in the laboratory are unable to yield an identification or they provide an unusual identification or one that conflicts with phenotypic characteristics. The use of artificial intelligence and machine learning has made its way into the clinical microbiology laboratory, largely due to the digitization of microbiology and increasing technology use.

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Mycobacterial inactivation protein extraction protocol for matrix-assisted laser desorption ionization time-of-flight characterization of clinical isolates

Abstract: The MIPE protocol produces a high quality protein extract with improved isolate identification without compromising result turn-around-times or laboratory safety.

Cited by 2 publications

References 2 publications

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol

Systems for Identification of Bacteria and Fungi

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