MALDI-TOF MASS SPECTROMETRY

• • Shift from Biochemical to MALDI-TOF MS Bacterial Identification
  • Matrix Assisted Laser Desorption Ionization (MALDI) is a very sensitive technique for determining the mass of proteins, peptides, or polymers. 
  • MALDI is attached to a time of flight (TOF) analyzer which measures time it takes for the molecules to travel a fixed distance.
• MALDI-TOF MASS SPECTROMETRY

  • Rapid and cost-effective identification of bacteria directly from isolated colonies and positive culture bottles based on protein biomarkers.
  • Protein biomarkers - highly expressed proteins responsible for housekeeping functions, such as ribomasal (16S) and transcription/translation factor proteins
• MALDI-TOF MASS SPECTROMETRY

  • Identification of microbes by MALDI-TOF MS is done by either:

  1. Comparing the peptide mass fingerprint (PMF) of the unknown organism with the PMFs contained in the database, 

  2. or by matching the masses of biomarkers of the unknown organism with the proteome database.
• MALDI-TOF MASS SPECTROMETRY

  • More complicated than Gene sequencing as this method looks at the protein biomarkers.
  • MALDI-TOF Mass is rarely used clinically; he mentions that he was only able to use it for research and it is not as user-friendly as the other techniques.
  • Mass Spectrometry (MS) creates a unique molecular fingerprint of an organism.
• MALDI-TOF MASS SPECTROMETRY

  • Measures highly abundant proteins that are found in all microorganisms and create measurable peaks.
  • The characteristic patterns of these highly abundant protein peaks are used to reliable and accurately identify a particular microorganism.
  • This is subjective and difficult to do in real life due to its comparative nature.
• MALDI-TOF MASS SPECTROMETRY

  • The pattern created is compared to a computer database to determine the identity of the microorganism.
  • Some softwares compares automatically, but in Prof. Ulanday’s experience, he had to manually do it to confirm the findings of the machine.
• MALDI-TOF Process

  • Phase 1: Ionization Step

  1. Initially, the samples are fixed in a crystalline matrix, in the initial ionization step, they are bombarded by a laser.
  2. The sample molecules vaporize into the vacuum while being ionized at the same time. 
  3. High voltage is then applied to accelerate the charged particles.
• MALDI-TOF Process

  • Phase 2: Time Of Flight - Mass Spectrometry Phase
  • Linear Mode

  1. Particles will impinge upon the linear detector within a few nanoseconds after ionization. 
  2. Higher mass molecules will arrive later than lighter ones. 
  3. Flight time measurements make it possible to determine molecular masses directly. Each peak in the spectrum corresponds to the specific mass of the particle along the time axis, starting with the ionization moment. 
• MALDI-TOF Process

  Phase 2: Time Of Flight - Mass Spectrometry Phase

  • Reflector Mode

  1. Particles are diverted so that they fly towards a second detector. In addition to extending the flight distance, the reflector also focuses the masses.

  • The combination of these two effects makes for higher resolution than in the linear mode. 
• MALDI-TOF MS Principle

  1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI target, which is usually a sample support. 

  • The sample for analysis (A) is prepared by mixing or coating with a solution of an energy-absorbent, organic compound called matrix (M). 
  • The mixture of sample and matrix crystallizes due to the vacuum environment.
• MALDI-TOF MS Principle

  • 2.Laser ionizes matrix molecules, making it dissipate. 
  • Upon a UV Laser pulse, there will be separation of the different molecules or ions.
  • A dry sample in a matrix solution absorbs the energy from a laser beam resulting in ionization of the sample solution. 
  • Desorption and ionization with the laser beam generates singly protonated ions from analytes in the sample.
  • That dissipation will then be measured by the time of flight (TOF) in the next steps.
• MALDI-TOF MS Principle

  • 3. All ions are accelerated to the same kinetic energy, enter the flight tube and are separated according to mass.
  • The protonated ions are accelerated at a fixed potential, where these separate from each other on the basis of their mass-to-charge ratio (m/z).
• MALDI-TOF MS Principle

  • 4. Ions are detected at the end of the flight tube.
  • The matrix, as it moves, the speed of the different ions are different (observe the location of the matrix ion and the protein biomarkers in the figure below). 
  • Speed is detected through a detector and it will form a mass spectrum, which is measured by mass-to-charge. 
• MALDI-TOF MS Principle

  • 5. The microorganism signature (e.g. the peaks) are the different matrix ions. Of importance are the red lines,  which are used to identify the MPF of the unknown organism with the PMFs contained in the database, or by matching the masses of biomarkers of an unknown organism with the proteome database.
• Ionization of Intact Proteins and Molecular Weight

  • Utilizes Peptide Mass Fingerprint
  • One of the disadvantages of the machine is that it is too big. It has to be in a tube so that the ions can still fly and be effectively measured.
• Drawbacks

  • Use of a chemical matrix (mixed with the sample)
  • Use of laser (used to effect desorption and ionization of the analyte)
• Solution

  • Use of electrospray ionization (ESI)-MS
  • Analyzes samples in a liquid state 
  • ionization is carried out at atmospheric pressure, without recurring to the same lasers as in MALDI-TOF-MS
  • Has a large spectra of applications regarding microbial identification
• CHOOSING THE RIGHT TEST (CLINICAL PERSPECTIVE)

  • Isolation and culture are possible as long as live bacteria are present in tissues,
  • After treatment of the bacteria that colonizes, it starts to wane and die.
  • Culture is more resource-intensive because of the need for space, culture media, and laboratories. 
  • Appropriate if you are looking for live organisms
  • If there is contamination through culture, we may see it based on the number of organisms present. However, for PCR, it is not as easy.
• CHOOSING THE RIGHT TEST (CLINICAL PERSPECTIVE

  • Bacterial DNA can be detected during the same period and also as far as dead microorganisms remain in tissues.
  • This is the advantage of DNA analysis or PCR, as they cannot distinguish live or dead organisms. As long as a remnant sequence of the dead organism is there, it will turn out positive.
  • It will be a disadvantage if the researcher is trying to detect live organisms because it may result in false positives. It also depends on what the researcher wants to know.
• Bacterial DNA can be detected during the same period and also as far as dead microorganisms remain in tissues.

  • It is possible for somebody to be positive for PCR but negative for culture. 
  • This is why the gold standard for TB testing is culture.
  • We want to identify live organisms, not just remnants because it is more clinically relevant.
  • Overly sensitive nature of PCR might be confusing for clinicians because that sensitivity may be due to contamination.
• • Specific antibodies appear during the clinical course of the disease and persist generally for months or years.
  • If you are looking towards the future, you can look into antibodies. 
• • Pathologic changes can be observed soon after the contamination and, in an acute infection, will decline rapidly after elimination of the bacteria.
  • If we want to do histology or look into the changes of the tissue, we have to get the sample during treatment because once the tissue is healed, histologic changes will not be apparent.