Introduction to MALDI-TOF

Mass Spectrometry
Principles of MALDI-TOF
Overview
• Matrix Assisted Laser Desorption/
Ionisation – Time of Flight (MALDI-TOF)
mass spectrometry used to detect and
characterize mixtures of organic
molecules.
• In micro used as a rapid, accurate and
cost-effective method for ID of
microbes
• 2 commercially available:
• Vitek MS (bioMerieux)
• MALDI Biotyper CA system (Bruker
Daltonics)

Bruker, MBT Compass User Manual, 2018

Mass Spectrometry
• Mass spectrometry (MS) measures mass-to-charge ratio (m/Q) of
ions.
• Results presented as a mass spectrum - a plot of the ion signal as a
function of the mass-to-charge ratio.
• Spectra used to determine
• elemental or isotopic signature
• the masses of particles
• the chemical ID or structure
Three components of MS
• An ion source
• Sample (solid, liquid, or gaseous) is ionized - for
solids via electrons or MALDI
• A mass analyzer
• Ions then separated based on mass-to-charge ratio
• Done by acceleration and subjecting to an electric or
magnetic field
• Ions with a lower mass will reach the detector first.
• A detector
• records either charge or current produced when ion
passes by or hits a surface
• produces a mass spectrum (mass-to-charge ratio)
• Usually an electron multiplier
MALDI – TOF MS
1. An ion source
• Laser and ionization chamber to ionize
sample and transfer into a gas phase
• Uses laser energy-absorbing matrix to
create ions from large molecules
2. A mass analyser
• separates ionized analytes according to
their mass (all same charge)
• TOF uses electric field to accelerate ions
& measure time to reach detector.
3. A detection device to monitor
separated ions
Patel, Clin Chem, 2015
Procedure
• Combine isolated colony
(analyte) , formic acid &
matrix on MALDI plate
• Solvents vaporize, leaving
only the recrystallized
matrix with analyte
embedded
Matrix
• Matrix isolates molecules from each other, protecting them from
fragmentation and enabling desorption by laser energy

• Consists of:
• Small crystalised acid molecules – usually sinapinic acid, alpha-cyano (alpha
matrix) or DHB acid.
• Purified water
• Organic solvent (alcohol or acetonitile)
• Trifluoroacetic acid 2.5%
Ion Source - Laser
• Uses UV lasers (nitrogen laser light, wavelength 337nm)
• Laser pulses fired at the matrix crystals in the dried-droplet spot.
• Matrix absorbs the laser energy converting it to an ionised state
• Charge is transferred to analyte (random collision in the gas phase)
• Ionised analyte and matrix molecules are desorbed from the plate

Shimadzu, Principles of MALDI-

TOF MS, 2020
Creative Proteomics, MALDI-TOF MS,
June 2020
Mass Analyser – Time of Flight
• Ionized microbial molecules accelerated
through a positively charged electrostatic field
into time of flight (TOF) tube
• Inside vacuum tube ions travel toward an ion
detector
• Small analytes travel the fastest (generating
mass spectrum)
• Ions emerge from the mass analyser and hit
the ion detector  generate a mass spectrum
representing the number of ions of a given
mass impacting the detector over time
Carroll, MCM 12 ed, 2019
Patel, Clin Chem, 2015
Shimadzu, Principles of MALDI-
TOF MS, 2020
Shimadzu, Principles of MALDI-
TOF MS, 2020
Results
• Mass spectrum provides profile unique to
individual types of microbes, with peaks
specific to genera and species
• Once acquired compared to a database of
reference spectra
• A value - percentage or score is produced
Results
Results
• ID is started immediately after mass
spectrum available.
• During the run the appearance of sample
and QC positions in the MALDI plate
display reflects the success of the
measurement and ID a each position
• If spectrum measurement successful - left
half of the sample is green.
• If measurement fails - left half of the sample
is orange
• Colouring of right half of sample position
indicates the score value of ID

Bruker, MBT Compass User Manual, 2018

Bruker, MBT Compass User Manual, 2018
Bruker, MBT Compass User Manual, 2018
 Consistency Description
MALDI Scores High (A) Best match is a high-confidence ID
Second-best match is:
• Higher the log (score),  high- confidence ID identical sp ID to best match
 low- confidence ID identical to genus in best match
higher the similarity  non-identification
between mass spectrum Low (B) Requirements for high consistency not met.
of isolate & the database Best match is a high- or low- confidence ID
Second-best match is:
entry in the reference  high- or low-confidence ID identical to genus in best
library match
 non-identification
None (C) Requirements for high or low consistency not met

Bruker, MBT Compass User Manual, 2018

Bruker, MBT Compass User Manual, 2018
Advantages
• Rapid (≤3 minutes per isolate)
• Inexpensive - low reagent cost
• Small amounts of organism are required
• Direct sample ID possible
• Reduced labour
• Accurate ID
Limitations
• Databases are proprietary unlike publicly available sequence databases
• Difficulties in ID with some organisms
• Difficulty analysing mixed cultures
• Identifying organisms from liquid cultures
• Low identification scores - repeat testing for 10% of isolates
• Growth on some media may be associated with low scores
• Small/mucoid colonies may fail ID
• ID of biosafety level 3/4 organisms
• Requires room temperature (20-25 °C)
• Human error

Carroll, MCM 12 ed, 2019

Difficulties in Identification
• Misidentification rare
• Can occur with closely related organisms
• E. coli and Shigella
• Discrimination between species from same complex eg. E cloacae complex
• Salmonella can only be ID to genus level - No typing
• Difficulties with some species - alpha haemolytic strep
• Mycobacteria & filamentous fungi
Considerations for other organisms
• Mycobacteria:
• Requires processing to kill tested bacteria, break down cell envelopes, disrupt
clumped cells
• Can ID most clinically relevant species
• MTB complex ID to complex level only
• Some related mycobacterium species not well differentiated (M. chimaera and M.
intracellulare)
• Enhanced databases ID Nocardia – often specific extraction processes
needed
• Fungi:
• Can identify yeast well
• Filamentous fungi limited - variable phenotypes & protein spectra vary with growth
conditions
• Available for aspergillus, fusarium & mucorales

Carroll, MCM 12 ed, 2019

Common Sources of Error
• Colony inoculation in erroneous target plate locations
• Testing impure colonies
• Smearing between spots
• Failure to clean target plates
• Entry of wrong results
Conclusion
• MALDI-TOF MS utilizes:
• Laser & matrix as an ion source
• TOF (electric field) as a mass analyser
• Ion detector

• Provides a rapid, accurate and cost-effective method for ID of many

bacteria & yeast

• Several limitations which operators need to be aware of when

reporting & troubleshooting