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

Methods for Experimental Investigation of Protein Structure
Electron Impact Mass Spectrometry
General Principles of Mass Spectrometric Amino Acid Sequence Determination

The main advantage of mass spectrometry in determining the Amino Acid Sequence of Peptides and Proteins is its ability to analyze mixtures of these substances. The classical dansyl-Edman method requires the mandatory preliminary isolation of individual fragments after Cleavage of the protein chain. This step is labor-intensive and limits the entire Edman sequencing process. In mass spectrometry, there is no need to isolate individual fragments; a rough Separation of the entire mixture into separate fractions is sufficient.

Due to the varying volatility of modified protein chain fragments, their individual components can be sequentially evaporated by gradual heating. With this approach, the gas phase is always enriched with a single specific fragment. By comparing mass spectra obtained at different temperatures, it is almost always possible to unambiguously determine The amino acid sequence of individual fragments. This technique yields acceptable results when analyzing mixtures containing up to five different peptides.

To increase the reliability of Conclusions regarding the order of Amino Acids in the original protein, mass spectrometry is typically combined with classical Methods, generally following this strategy.

> Determine the N-terminal amino acid using an automated Sequencer or manually.

> Analyze the peptides formed after specific Enzymatic cleavage of the protein with Trypsin or Chymotrypsin using the Edman method.

> Determine the amino acid sequence of short peptides obtained by nonspecific Protein Cleavage with Elastase or subtilisin using mass spectrometry in mixtures. Combined with step 2, this makes it possible to establish the insertion sites of large peptide fragments within the protein chain.

The methodology for exclusively mass spectrometric protein sequencing includes the following steps.

> Cleave the protein nonspecifically with elastase or subtilisin into relatively small fragments.

> Separate short peptides from the unhydrolyzed protein and large fragments by Gel filtration.

> Separate the mixture of short peptides by cation-exchange Column Chromatography (e.g., DOWEX) or High-Performance Liquid Chromatography. When using cation exchangers, the best eluent is a pyridine-acetic acid mixture, whereas for HPLC, it is an acetic acid-propanol mixture.

> Collect the fractions containing up to 5 peptides during chromatography and dry them down.

> Perform chemical Modification of the peptides in the obtained fractions and determine their amino acid sequence using mass spectrometry.