Basic Principle of MALDI-TOF Mass Spectrometry

The presentation titled **"Basic Principle of MALDI-TOF Mass Spectrometry"** by **Shafiya Sakina** explains the **MALDI-TOF MS** technique, a soft ionization method used in mass spectrometry to analyze biomolecules such as **DNA, RNA, proteins, peptides, and sugars**. **MALDI** stands for **Matrix-Assisted Laser Desorption Ionization**, and **TOF** stands for **Time of Flight**. The process separates ions based on their mass-to-charge ratio by measuring the time taken to reach a detector.

The presentation details the **history of MALDI-TOF MS**, tracing its discovery to **Franz Hillenkamp, Michael Karas (1985), and Koichi Tanaka (1988)**, who coupled TOF analysis with MALDI for macromolecule study, eventually earning the **2002 Nobel Prize in Chemistry**.

The **MALDI-TOF process** is divided into two phases: **Ionization Phase** and **Time of Flight Phase**, with the latter having **Linear Mode and Reflector Mode**. The ionization phase involves laser bombardment of a **crystalline matrix-embedded sample**, causing vaporization and ionization. The TOF phase then separates ions based on their mass, with heavier molecules arriving later in **linear mode**, while **reflector mode** improves resolution by refocusing ions.

The **procedure** involves dissolving the analyte in a solvent, mixing it with a matrix, crystallizing the mixture on a metal plate, and analyzing it in the MALDI-TOF instrument. **Formic acid** is used for better identification of bacterial species, particularly **Gram-positive and sugar non-fermenting bacteria**.

The presentation also highlights **applications** of MALDI-TOF MS in nucleic acid analysis, disease diagnosis, pathogen identification, small molecule analysis, microbial species identification, and bloodstream infection diagnosis.

1. Basic principle of MALDI-TOF Mass spectrometry BY- SHAFIYA SAKINA

2. MALDI-TOF MS MALDI is a soft ionization technique used in mass spectrometry, alllowing the analysis of biomolecules (DNA, RNA, proteins, peptides and sugar or polymers). Also used for peptide mass fingerprinting. MALDI stands for Matrix-Assisted Laser Desorption ionization . TOF stands for Time Of Flight . A method that separates ions by their mass to charge ratio and determines this  ratio by the time it takes for the ions to reach a detector. It is three step method-: The sample is mixed with suitable matrix and applied to a metal plate. A pulsed laser irradiate a sample tiggering desorption of matrix material. Ionization of analyte molecules.

3. In 1985, the concept of MALDI MS was originally discover by Franz hillenkamp , Michael Karas and their colleagues. In 1988, mass analyzer time-of-flight (TOF) was coupled to MALDI by Koichi Tanaka and his colleagues for analysis of macromolecules, specially for proteins. After that it start using in varity of molecules and being awarded by the Nobel Prize in 2002 in chemistry. MS HISTORY Koichi Tanaka(2002)

4. Basic principle of MALDI-TOF The MALDI-TOF process is a two phase procedure; 1.Ionization phase 2.Time of flight phase The second phase has two modes; 1.Linear mode 2.Reflector mode

5. Ionization phase Initially, the sample are fixed in a crystalline matrix on a target plate and are bombarded by a l aser. The sample molecule vaporize into the vaccum while being ionized at the same time. High voltage is then applied to accelerate the charge particles. FIG; Sample preperation and deposition at sample plate

6. Time Of Flight Phase The second step is the time of flight mass spectrometry phase having two modes; Linear mode Higher mass molecules will arrive later than lighter ones. Flight time measurement makes it possible to determine molecule masses directly. Each peak in the spectrum corresponds to the specific mass of the particle along the time axis,starting with ionization moment. Reflector mode The particles are diverted so that they fly towards a second detector. In addition to extending the flight distance, the reflector also focuses on the masses. The combination of these two effects makes for higher resolution than the linear mode The net result is compared with those of well characterized organism available in the reference library database to identify the isolate.

7. The instument can be operated either linear mode or reflectron mode Fig;linear vs reflectron mode in MALDI-TOF

8. PROCEDURE  First, the analyte should be dissolved in a solvent making up to 0.1mg/ml and the matrix should be dissolved with a saturated or concentrated solution of about 10mg/ml. Both the solution is then mixed together in 1000:1 to 100,000:1 ratio. 01 The mixture is placed on a metal target plate which crystallizes on drying and forms a solid deposit. Then the mixture is transferred into the MALDI – TOF instrument for analysis. 02 Sublimation and ionization separate the ions depending upon the size and charge ratio through a TOF analyzer which is operated on the MS software 03 To increase the ability in identifying gram-positive and sugar non fermenting bacterial species, formic acid is used with the preparatory extraction of microbes whereas gram- negative bacteria can be identified using direct cell profiling 04

9. Process of mass spectrometry

10. Applications Nucleic acid analysis Disease diagnosis Pathogen identification Small molecule analysis Microbial species identification Blood stream infection diagnosis .

11. References en.wikipedia.org/wiki/Matrix- assisted laser-desorption/ionization http;//pubs.acs.org/ doi /10.1021 http;//microbeonline.com/ maldi - tof - ms -principle-applications-microbiology/

12. Thank You