Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in Clinical Microbiology
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Robin Patel, M.D., is the Division Chair of Clinical Microbiology in the Department of Laboratory Medicine and Pathology in Rochester, Minnesota. She holds the academic rank of Professor of Medicine and MMicrobiology.
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Transcript and References
Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. In this month’s "Hot Topic," Dr. Robin Patel, discusses how matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) works for bacterial identification, including the strengths and limitations of this technology, and Mayo Clinic’s experience with the technology in the clinical laboratory. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patient’s health care.
Thank you for the introduction Dr. Binnicker.
Dr. Patel reports grants from CD Diagnostics, BioFire, Curetis, Merck, Contrafect, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, EnBiotix, Contrafect and The Medicines Company. Dr. Patel is or has been a consultant to Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest, Heraeus Medical, and Qvella; monies are paid to Mayo Clinic. In addition, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. Dr. Patel receives travel reimbursement from ASM and IDSA and an editor’s stipend from ASM and IDSA, and honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course.
The objectives of this presentation are: 1. to explain how matrix-assisted laser desorption ionization time of flight (also referred to as MALDI-TOF) mass spectrometry works as a clinical microbiology laboratory application, 2. to explain the role of MALDI-TOF mass spectrometry in clinical microbiology, and 3. to highlight our experience with MALDI mass spectrometry.
How Does Mass Spectrometry Work?
Mass spectrometry measures particles based on their mass-to-charge ratio. To do this, a sample (in the described method, the whole organism, either a bacterium or a fungus) is exposed to an ion source, and its particles (in the described methods, proteins) are ionized, separated based on their mass-to-charge ratio, detected, and then the generated mass spectrum (in the described method) is compared against a library of mass spectra.
Types of Mass Spectrometry
Mass spectrometry requires an ion source, mass analyzer, and detector. There are multiple possible ion sources. In the remote past, ionization required molecules in the gas phase, limiting analysis to volatile compounds or those that could be rendered volatile. Large nonvolatile polar molecules, such as proteins, were not easily analyzed and, therefore, mass spectrometry was not used for protein analysis. With the arrival in the late 1980s of matrix-assisted laser desorption ionization, mass spectrometry based on microbial proteomics has become possible. MALDI is a soft ionization technique allowing molecules to remain relatively intact during ionization. Large proteins can be measured as little protein fragmentation occurs. Following ionization, the ions are separated, enabling measurement of mass. Using the approach covered in this presentation, ions are separated by time of flight in a flight tube.
MALDI-TOF Mass Spectrometry
I will first go over how MALDI-TOF mass spectrometry is practically done in the clinical microbiology laboratory, starting from a colony. Commercial systems for clinical microbiology laboratories are available from bioMérieux, Inc. and Bruker Daltonics, Inc. The example shows the latter because it is the system with which I have had the most experience.
A colony may be picked directly from a plate and smeared onto a target plate. Then, 1 to 2 µL of a “matrix” consisting of, for example, alpha-cyano-4-hydroxycinnamic acid dissolved in acetonitrile and trifluoroacetic acid, is added and dried on the plate.
Some organisms require preparatory extraction to generate spectra of sufficient quality to enable microbial identification. For example, the isolate of interest may be placed into a microcentrifuge tube with 70% ethanol, mixed, and centrifuged, with the supernatant decanted, the pellet dried, 70% formic acid and acetonitrile added, and the mixture vortexed and centrifuged again. The supernatant can then be deposited onto a target plate, dried, and overlain with matrix.
Alternatively, on-plate formic acid treatment can be performed; this is also referred to as “on-plate extraction” or “extended direct transfer.” Using this approach, whole cells from colonies are moved to the target plate and then exposed to a formic acid solution, either by adding the formic acid solution prior to colony transfer or by overlaying the transferred colony with formic acid solution. This is then dried, overlain with matrix, and the process continued.
MALDI-TOF Mass Spectrometry: Bruker Biotyper
The target plate is placed into the plate chamber of the mass spectrometer, the plate chamber is closed, and analysis is performed. Target plates have multiple spots, so multiple isolates can be prepared and analyzed together, at about 2 to 3 minutes per sample.
Matrix-Assisted Laser Desorption Ionization
Let us look at the details. The sample is mixed with the matrix and co-crystalized onto the target plate (the “matrix-assisted” component of matrix-assisted laser desorption ionization). The matrix “buffers” the sample, preventing its decomposition and enabling transformation of laser light into heat.
Matrix-Assisted Laser Desorption Ionization
A laser is applied (the “laser” component of matrix-assisted laser desorption ionization).
Matrix-Assisted Laser Desorption Ionization
The matrix absorbs energy from the laser, releasing it into the sample as heat. This causes the sample to desorb and form singly charged ions (the “desorption ionization” component of matrix-assisted laser desorption ionization).
Time of Flight
Next, the mass of the ions is analyzed. This is accomplished using a flight tube, the lighter ions traveling faster and, therefore, being detected earlier than the heavier ions. In the described method, particles are typically singly charged. The net result is generation of a mass spectrum in which the mass-to-charge is plotted against signal intensity. Only highly abundant proteins that are of low mass and ionize readily are detected. These are typically ribosomal proteins, although the specific nature of the analyzed proteins is not part of the analysis.
Mass-Spectrum Generated Compared with Library (Database)
The mass profile is used as a fingerprint or mass spectrum (as shown on this slide) to compare with those of well-characterized organisms in a database. The spectrum typically includes genus- and species-specific peaks so that with a comprehensive library of spectra, the genus and often the species of the organism is determined using bioinformatics.
Laboratory Workflow of the Past
Matrix-assisted laser desorption ionization time of flight mass spectrometry has changed our workflow, which historically involved rapid biochemicals, an automated phenotypic identification system, long-tubed biochemicals, and 16S ribosomal RNA gene sequencing . . .
Laboratory Workflow Since 2011
. . . to a matrix-assisted laser desorption ionization time of flight mass spectrometry-based approach, which typically abrogates the need for many other identification tools traditionally used.
The next few slides show examples of the types of organisms that can be identified with MALDI-TOF mass spectrometry based on the current FDA-approved/cleared systems.
2 Slides: Aerobic Gram-Positive Bacteria
There are many aerobic Gram-positive bacteria that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
Gram-Negative Bacteria, Enterobacteriaceae
There are multiple Enterobacteriaceae that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems. Note that the Escherichia coli and Shigella species cannot be differentiated.
Gram-Negative Bacteria, Non-Enterobacteriaceae
There are also many Gram-negative bacilli that are not Enterobacteriaceae that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
Fastidious Gram-Negative Bacteria
There are several fastidious Gram-negative bacteria that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
There are multiple anaerobes that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
Mycobacteria and Nocardia Species
There are numerous mycobacteria and Nocardia species that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
Additionally, there are various yeasts that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
Filamentous Fungi FDA-Approved/Cleared on Vitek MS System
And finally, there are filamentous fungi that are FDA-approved/cleared on the Vitek MS system.
Beyond FDA-Approved/Cleared Systems
Bruker also has a research use only (RUO) database, as well as other RUO databases, including a filamentous fungi library, a mycobacteria library, and a “security relevant” database. bioMérieux likewise has an RUO database called VITEK MS RUO. In addition, users can construct their own databases, as we have done at Mayo Clinic.
MALDI-TOF Mass Spectrometry: Strengths
Matrix-assisted laser desorption ionization mass spectrometry has a number of strengths. It is automated, green, it doesn’t require specific expertise in mass spectrometry, and it has a rapid turnaround time and high throughput capability. It only requires a single colony and is associated with a low exposure risk due to sample inactivation. Although not covered in today’s presentation, this approach is cost-effective and has demonstrated high inter-laboratory reproducibility. It has broad applicability (covering all types of bacteria, including anaerobes as well as fungi). Finally, the system is open and adaptable by the user.
MALDI TOF Mass Spectrometry: Limitations
There are limitations to matrix-assisted laser desorption ionization mass spectrometry. No susceptibility information is provided, and the technology is not generally useful for direct testing of clinical specimens. Some organisms require repeat analyses and additional processing. The acceptable score or percentage cutoffs for identification of genera and species, which were not covered in this presentation, vary between studies. Some closely related organisms are not differentiated. Comparison of data from the two companies’ instruments is not feasible. Laboratories acquiring the needed equipment will suffer financial loss on existing equipment. And finally, instrument downtime can be problematic if institutions only have single systems.
- Saffert RT, Cunningham SA, Ihde SM, et al. Comparison of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometer to BD Phoenix automated microbiology system for identification of Gram-negative bacilii. J Clin Microbiol. 2011 Mar;(49)3:887-892.
- Alatoom AA, Cunningham SA, Ihde SM, et al. Comparison of direct colony method versus extraction method for identification of Gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2011 Aug;49(8):2868-2873.
- Alatoom AA, Cazanave CJ, Cunningham SA, Ihde SM, and Patel R. Identification of non-diphtheriae Corynebacterium by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012 Jan;50(1):160-163.
- Marko DC, Saffert RT, Cunningham SA, et al. Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of nonfermenting gram-negative bacilli isolated from cultures from cystic fibrosis patients. J Clin Microbiol. 2012 Jun;50(6):2034-2039.
- Theel ES, Schmitt BH, Hall L, et al. Formic acid-based direct, on-plate testing of yeast and Corynebacterium species by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012 Sep;50(9):3093-3095.
- Schmitt BH, Cunningham SA, Dailey AL, Gustafson DR, and Patel R. Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation. J Clin Microbiol. 2013 Mar;51(3):782-786.
- Patel R. Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology. Clin Infect Dis. 2013 Aug;57(4):564-572.
- Patel R. MALDI-TOF mass spectrometry: Transformative proteomics for clinical microbiology. Clin Chem. 2013 Feb;59(2):340-342.
- Cunningham SA, Mainella JM, and Patel R. Misidentification of Neisseria polysaccharea as Neisseria meningitidis with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2014 Jun;52(6):2270-2271 .
- Mushtaq A, Chen DJ, Strand GJ, et al. Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles—a retrospective chart review. Diagn Microbiol Infect Dis. 2016 Jul;85(3):372-376.
- Carroll K and Patel R. Systems for identification of bacteria and fungi. Manual of Clinical Microbiology. 1. 12 ed: ASM Press; In Press.