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.
The objectives of this presentation are 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, to explain the role of matrix-assisted laser desorption ionization time of flight
mass spectrometry in the clinical microbiology,
and to highlight our experience with matrix-assisted laser
desorption ionization time of flight mass spectrometry.
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.
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.
Matrix-assisted laser desorption ionization 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.
I will first go over how matrix-assisted laser desorption ionization time of flight 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, of
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.
The target plate is placed into the plate chamber of the mass spectrometer, the plate
chamber is closed, and analysis performed.
Target plates have multiple spots, so multiple isolates can be prepared and
analyzed together, at about 2 to 3 minutes per sample.
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.
A laser is applied, (the “laser” component of 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).
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.
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.
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…
…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 matrix-assisted
laser desorption ionization time of flight mass spectrometry, based on the current FDA-
approved/cleared systems.
There are many aerobic Gram-positive bacteria that are FDA-approved/cleared on the Vitek
MS and MALDI Biotyper CA systems.
There are multiple Enterobacteriaceae that are FDA-approved/cleared on the Vitek MS and
MALDI Biotyper CA systems.
Note that Escherichia coli and Shigella species cannot be differentiated.
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.
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.
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.
And finally, there are filamentous fungi that are FDA-approved/cleared on the Vitek MS system.
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.
Matrix-assisted laser desorption ionization time of flight 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.
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.