This document provides a summary of proteomic techniques, such as mass spectrometry. The focus lies on tandem MS, peptide fragmentation, MS/MS, Maldi and electrospray, Time-of-flight, orbitrap and the pathology of Alzheimer’s disease and how the MS contributes to the research. There is also a bri...
Experimental Cell Biology II: proteomics/mass spec
Proteomics Tandem mass spectrometer
Proteomics relies on the use of mass A tandem MS is a device that uses two mass
spectrometry. A mass spectrometer is a device spectrometers. First, the protein is digested and the
that measures the mass of analytes such as peptides are injected into the mass spectrometer.
proteins, and peptides, but also carbohydrates, Then the peptides go through the first MS to measure
fats, small molecules and chemicals. When a the mass and charge of the peptides. After this is
molecule gets injected into the MS, a detector done, you can select a peptide from the mass
gives a peak that stands for the mass and spectrum that goes through the second MS. The
peptide is ionized and fragmented by energy in the
intensity of the molecule. This peak intensity
fragmentation chamber. This results in random
correlates to the peptide concentration,
fragments of the peptide. The MS then measures the
therefore you can quantify the number of mass and charge of these fragments and can be sued
peptides in your sample. to deduce the amino acid sequence.
A protein consists of amino acids and each Peptide fragmentation
amino acid consists of peptides that each have Each amino acid mass is already known, so by
a specific mass. subtracting the masses of the fragmented ion
peptides the sequence of the peptide can be revealed.
• MS measurement of peptides is excellent
Sometimes some of the fragments go missing during
• MS measurement of proteins is possible, but
fragmentation, which leads to difficulty in finding the
with low resolution and poor mass accuracy. amino acid sequence. Once the fragmentation
spectrum is revealed the amino acid sequence is
Before the analysis, a protein has to be calculated using a database according to their
digested into peptides using an enzyme called corresponding masses.
trypsin, since this is more accurate and gives a
higher resolution. The enzyme cleaves specific
peptide bonds between the carboxyl group of
arginine or the carboxyl group of lysine and
the amino group of the neighbouring amino
acid. The peptides are derived from the protein
and the peptides can be identified.
Nowadays a sample could consist of more than 3000
proteins that generate about 30 peptides, which in
this case is about 90.000 peptides. This can not be
injected into the MS right away, but the complexity
Though, there are peptides that have the same
has to be reduced by high-pressure liquid
mass. Therefore, they need to determine the chromatography (HPLC) or SDS-PAGE where they
amino acid sequence for the high specificity of can cut the gel into several pieces and isolate the
peptide or protein identification. proteins.
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