How Does a Mass Spectrometer Sequence Amino Acids After Fragmenting Peptide Chains? Shouldn’t It Only Identify the Termini
A mass spectrometer is a powerful tool used to determine amino acid sequences. This technique typically involves fragmenting peptide chains into smaller pieces through methods such as collision-induced dissociation (CID) or electron transfer dissociation (ETD), and subsequently deducing the amino acid sequence of the original peptide by measuring the masses of these fragments.
The key steps in mass spectrometer analysis include:
1. Ionization
Initially, the sample is ionized, commonly via electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI).
2. Fragmentation
Subsequently, the ions are fragmented within the mass spectrometer. Fragmentation can occur through various mechanisms, including CID or ETD.
3. Measurement
Finally, the mass-to-charge ratios (m/z) of the resulting fragment ions are measured. These values enable the deduction of the amino acid sequence of the original peptide.
In this process, the amino acid sequence of the original peptide can be inferred by comparing the mass differences between fragment ions. For instance, if one fragment ion has a mass of 1000 and another has a mass of 1200, the difference of 200 mass units can be attributed to one or more amino acids. Reference tables can be used to identify the amino acid(s) corresponding to this mass difference.
Beyond identifying the termini, it is possible to determine amino acid sequences at internal positions. Mass spectrometer analysis of peptide segments generates a series of fragment ions of varying sizes, primarily resulting from cleavages at peptide bonds. These fragments include b-ions, counted from the N-terminus, and y-ions, counted from the C-terminus. By correlating these two types of ions, the amino acid sequence of the original peptide segment can be determined.
For example, if a b-ion exhibits a mass difference relative to the intact peptide corresponding to the mass of a specific amino acid, and a y-ion also exhibits a mass difference matching that amino acid, this provides evidence that the peptide contains that amino acid at the respective position.
Thus, mass spectrometer sequencing allows the identification of not only terminal amino acids but also amino acids at internal positions of the peptide segment. This process involves complex data analysis, but specialized software such as SEQUEST, MASCOT, and MaxQuant facilitates interpretation of intricate mass spectrometry data and comparison with established protein databases to aid in the determination of potential amino acid sequences.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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