Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Electron Impact Mass Spectrometry
Requirements for Peptides Analyzed by Mass Spectrometry

Low-resolution Electron Impact Mass Spectrometry is widely used to determine the Amino Acid Sequence of acetylated or methylated Peptides. Mass spectrometers employed for peptide sequencing must meet two core requirements: they must provide high sensitivity and be capable of detecting ions with masses up to 1000 Da.

The length of peptides amenable to analysis by electron impact mass spectrometry is determined primarily by their volatility and the mass-to-charge ratios of the resulting ions. Typically, the number of amino acid residues in a peptide should not exceed ten. For peptides consisting of small, hydrophobic amino acid residues (such as Glycine, valine, and Alanine), 20-mer fragments (oligomers) may also possess sufficient volatility. Larger peptides can only be successfully analyzed by mass spectrometry if they undergo thermal fragmentation into smaller, highly volatile fragments within the ionization chamber of the spectrometer.

Sample size. The amount of peptide required for Sequence Determination by mass spectrometry depends mainly on its size, polarity, and fragmentation behavior. For medium- and high-grade commercial mass spectrometers (such as the KRATOS MS-50 or the VG Analytical ZAB), 30–50 nmol of a 10-residue peptide per analysis is generally sufficient.

Sample purity. The current strategy for peptide sequencing via mass spectrometry has been developed specifically for the analysis of mixtures. Consequently, the tolerance for contaminating peptides in the sample is no stricter than in the classical dansyl-Edman method. However, the presence of non-peptide impurities—such as salts, vacuum grease residues, and plasticizers—is strictly unacceptable in mass spectrometric analysis. For instance, even trace amounts of organosilicon compounds from silicone grease will almost completely suppress the electron impact ionization of peptides.



Last update: 06/08/2026

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