32.17:
Peptide Identification Using Tandem Mass Spectrometry
Unlike MALDI-TOF, tandem mass spectrometry, or MS/MS, employs two mass analyzers placed in series, separated by a collision cell. This technique can identify the amino acid sequence of a peptide from a complex mixture for detailed analysis.
First, an electrospray ionizer generates peptide ions and accelerates them to the first mass analyzer like a quadrupole. This analyzer acts as a mass filter allowing selection of a single peptide ion called a precursor ion of a specific mass-to-charge ratio.
Once inside the collision cell, the selected peptide ion collides with the gas molecules, primarily breaking the peptide bonds, and generating smaller ions.
These ions move into the second mass analyzer like a time of flight that separates ions based on their mass-to-charge ratio.
The obtained product-ion spectrum helps identify the peptide's amino acid sequence.
Combining MS/MS with liquid chromatography aids in continuous peptide analysis from complex biological samples.
MS/MS can also characterize lipids, identify post-translational protein modifications, and detect low levels of disease-specific biomarkers in biological samples.
32.17:
Peptide Identification Using Tandem Mass Spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of proteomics.
The different peptides obtained after enzymatic digestion can be further separated as much as possible as per the physical size and/or chemical properties using sophisticated instruments like gel electrophoresis or liquid chromatography.
This first stage of MS/MS allows the peptides to separate based on their mass-to-charge ratio, followed by breaking or fragmenting the selected peptide ion in the collision cell. The second mass analyzer helps build the fragmentation pattern to determine the sequence or identify the protein.
Studying the results and deducing the peptide sequence is extremely important once the spectrum is obtained. For this, numerous protein database search algorithms and bioinformatics tools help in sample elucidation from the obtained spectrum. In the case of an unknown protein, the obtained spectrum shows numerous overlapping fragments. However, as the spectral pattern is unique for a given protein, the analysis software compares the obtained spectrum with a database of known peptide sequences, thus elucidating the unknown protein from the overlapping fragments.
Different analyzer combinations can be used to create hybrid MS/MS instruments and thus increase the sensitivity of the results. For example, quadrupole time-of-flight (QTOF) is a combination of quadrupole and time-of-flight mass analyzers. The triple quadrupole mass spectrometer contains two quadrupole mass analyzers separated by a non-mass resolving quadrupole collision cell for fragmentation.
In newborn screening, the tandem MS uses dried blood-spot samples to help in a comprehensive assessment of inborn metabolic disorders like phenylketonuria, sickle cell disease, etc. The early identification helps substantial improvement in health outcomes.
Suggested Reading
- Mittal, Rama Devi. "Tandem mass spectroscopy in diagnosis and clinical research." Indian Journal of Clinical Biochemistry 30, no. 2 (2015): 121-123.
- Glish, Gary L., and David J. Burinsky. "Hybrid mass spectrometers for tandem mass spectrometry." Journal of the American Society for Mass Spectrometry 19, no. 2 (2008): 161-172.