Biological Analysis

Application of MALDI-TOF

MALDI-TOF/TOF mass spectrometer is an analytical instrument used in the field of biology. The first step of mass spectrometry analysis is to ionize the substance to be analyzed. For macromolecular substances such as proteins and peptides, some ion sources, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) will fragment macromolecular ions during the ionization process to form fragments.

The emergence of MALDI-TOF solves this problem, by referencing matrix molecules, producing no or fewer fragment ions. Its mild ionization conditions make it ideal for the determination of macromolecules such as proteins, peptides, biopolymers and DNA. MALDI-TOF ionizes the analyte molecules by introducing a matrix. The most common form of MALDI ionization is that the analyte molecules have a positive charge. After the gaseous analyte ions are generated, their mass is analyzed by time-of-flight mass spectrometry.

Main functions of MALDI-TOF

1. High-throughput proteomics: suitable for protein peptide mass fingerprinting, peptide fragment sequence tandem mass spectrometry (MSMS) analysis, unknown protein sequencing, protein post-translational modification identification, protein interaction; proteins, peptides, nucleic acids, oligosaccharides Molecular weight determination of biological macromolecules, etc.
2. Biomarker and quantitative proteomics research: suitable for quantitative analysis of protein differential expression and identification and analysis of disease-related markers. It is an ideal platform for isotope labeling-based relative quantification experiments, such as the iTRAQ reagent 8-Plex technology solution, which can achieve ideal results without additional hardware adjustments or software additions.​​
3. Biological tissue mass spectrometry imaging analysis.

MALDI-TOF application

Clinical diagnostic laboratories rely primarily on traditional phenotypic methods for diagnosing infections, and sometimes genetic sequencing techniques. Advances in technology include matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that has entered daily microbial practice. MALDI-TOF mass spectrometry produces specific mass spectral fingerprints that can be seen as unique features of microorganisms that help to accurately identify genus and species levels—with great potential for strain typing.

Bacterial identification:

Several methods utilizing MALDI-TOF mass spectrometry are now being used in microbiological diagnostic laboratories. One method compared the results with a commercially available mass spectrometry signature database to identify sample bacteria, and the second method used a proteome database to identify biomarker masses in bacteria from sequenced genomes. The second bioinformatics approach allows for changing differences in culture growth and sample processing conditions, while the first approach is particularly useful in routine laboratory methods, such as diagnostics, and can distinguish species from subspecies.

Given its accuracy, the technique can also be directly applied to a variety of clinical samples, most notably blood, cerebrospinal fluid, urine, pleural fluid, and peritoneal fluid. Due to the detection limitations of the current MALDI-TOF protocol, the main limitation is the number of bacteria present in the sample. To circumvent this disadvantage, large blood and urine samples are often required, as well as the use of cultures as an additional enrichment for blood.

The results of bacterial identification using MALDI-TOF mass spectrometry using either of the two methods described above, the diagnostic yield and accuracy largely depend on the bacterial taxonomy and the quality of the database used.

One of the main advantages of using the MALDI-TOF technique to identify bacteria is the rapid availability of results, usually ready in less than an hour. Furthermore, MALDI-TOF mass spectrometry enables precise identification of a large number of bacteria with scarce phenotypic features and requiring 16S rRNA gene sequencing prior to the MALDI-TOF era.

Fungal identification:

MALDI-TOF mass spectrometry was rapidly and successfully used for fungal identification. Currently, this method is mainly used for routine yeast identification, but further development (especially in sample preparation protocols and databases) is required to apply this method to other fungal groups such as dermatophytes and filamentous fungi.

Similar to the case of bacteria, misidentification or non-identification of fungal genera and species by MALDI-TOF mass spectrometry is essentially due to erroneous, missing or incomplete reference spectra in databases. The disadvantage is that the reference spectra currently contained in the commercially accessible database of MALDI-TOF mass spectrometry systems are incomplete.

Mass spectrometry identification MALDI-TOF advantages:

Compared to traditional methods of microbial identification, MALDI-TOF mass spectrometry significantly reduces engineer/technician working time (preparing samples) and turnaround time (obtaining results through automated analytical procedures) in most cases.

Mass spectrometry identification applications:

MALDI-TOF MS is used in a variety of industries including biopharmaceuticals, organic chemistry, metabolomics and genomics, as well as clinical and diagnostic and therapeutic applications. In organic chemistry applications, MALDI-TOF is used to analyze nucleic acid, protein, and polymer mass, and to identify complex mixtures of oligonucleotides and small proteins, providing useful information to biochemical and chemical researchers. MALDI-TOF MS plays a vital role in facilitating rapid patient diagnosis and improving health outcomes. A prime example of this is its use for routine classification of microorganisms in patient samples for clinical microbiology.

In conclusion

In conclusion, MALDI-TOF mass spectrometry is a fascinating new technique for microbial identification that is fast, efficient, cost-effective and easy to use.

AxisPharm Maldi-TOF Analysis Service

AxisPharm Laboratories is equipped with a Voyager DE MALDI-TOF system made by Applied Biosystems. We are routinely using this instrument in analyzing polymers, oligonucleotides, peptides, proteins, and many other macro-molecules that would be difficult or not possible to be detected with API sources or any other mass spec techniques. Masses of peptides (low molecular weight, 750-4,500) can be determined on low picomol quantities with an average mass accuracy about 0.2%. Under optimum conditions, the limit of sensitivity of tryptic peptides (below 4,000) is in the lower femtomol range. Masses can potentially be obtained on numerous biopolymers including oligosaccharides, nucleotides and proteins that range from ~600 to 750,000 Daltons.

Maldi-TOF Analysis