Written by María Gerpe, PhD

August 13, 2021

Introduction

Amino acid sequencing is commonly performed using Edman degradation or mass spectrometry (MS). While mass spectrometry is favoured for its high throughput capabilities and ease of use, both techniques possess their own features and limitations. This article summarizes some of the key pain points inherent in the two methodologies when determining the amino acid sequence.

Edman Degradation Procedure

Developed in the 1950’s, Edman degradation involves the cleavage of amino acids from a peptide or protein, followed by their stabilization and identification using electrophoresis or chromatography. The stepwise process is repeated until the entire amino acid sequence is resolved1.

This method is often reserved for specific conditions, when its molecular approach is especially effective. Otherwise, Edman degradation faces criticism for being a slow process (around 1 hour per cycle), limited in its ability to process peptides above 30 residues, and requiring relatively high quantities of peptide (100 pmol) to perform an analysis. Moreover, as Edman degradation only proceeds on samples of single proteins, it lacks high-throughput capabilities compared to other techniques, such as mass spectrometry, favored when samples are numerous. This remains a significant limitation of Edman sequencing, despite being partly assuaged in 1967 by automation and increased process efficiency of the process relative to a manual approach1.

In addition, due to the mechanism that Edman degradation employs, amino acid sequencing is not readily available when the protein of interest lacks a free α-amino acid group. Termed N-terminal blockage, this can result from co- or post-translational modification (e.g., by acetylation) and occurs in up to 50% of eukaryotic proteins. Fortunately, by subjecting the separated protein to enzymatic or chemical cleavage to generate shorter peptide fragments—which can then be isolated and sequenced—this issue can be resolved1.

Mass Spectrometry

The development of mass spectrometry heralded a new frontier of amino acid sequencing that brought about the decline in use of Edman degradation. MS typically involves the enzymatic digestion of proteins, followed by ionization of the resulting peptides, separation according to their mass-to-charge ratios, and detection using an ion detector.

A major pain point of amino acid sequencing associated with the bottom-up MS approach mentioned above, is the requirement of thoroughly annotated sequence libraries, where searches are only able to match fragments of entire proteins. Because proteins need to be digested into peptides around 5-20 amino acids in length prior to identification, and because some genomes have yet to be sequenced, some proteins sequenced via mass spectrometry can be misidentified. To avoid the challenges posed by reference libraries, algorithms can be designed to automatically identify the most likely candidates matching the protein of interest2.

Upon successful identification of the peptide, difficulty remains in discovering isoforms and assigning post-translational modifications. Often enrichment strategies, such as chromatography and ion exchange, are used to locate and decipher PTMs, however, this strategy is challenging to execute.

In the past, MS techniques suffered from low sensitivity. However, advances through the years have ensured sensitivity issues are improved for amino acid sequencing. MS can be coupled with assays like antibody-based enrichment to offer up to 100,000 times improved sensitivity. In addition, concerns surrounding sensitivity can be addressed using a nanopore; an analytical tool with extreme sensitivity.

A better alternative is the use of de novo protein sequencing by liquid chromatography tandem mass spectrometry. The latter technology does not rely on databases. Using data from the mass spectrometer instruments and machine learning algorithms, the protein sequence can be deciphered, bypassing the shortcomings mentioned in the above paragraphs. However, this technology is not widely available as many laboratories worldwide do not fully integrate machine learning capabilities with wet bench lab processes2.

Summary

Finding the correct technique for amino acid sequencing can be challenging. Edman degradation and mass spectrometry are two popular methods with each one possessing specific pain points. De novo protein sequencing by tandem mass spectrometry is a great tool to bypass these pain points. The latter type of amino acid sequencing technology continues to facilitate antibody discovery and development. Want to learn more? Contact a member of the Rapid Novor team today.

References

1 Alfaro, J. A. et al. The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604-617, doi:10.1038/s41592-021-01143-1 (2021).

2 Hughes, C., Ma, B. & Lajoie, G. A. De novo sequencing methods in proteomics. Methods Mol Biol 604, 105-121, doi:10.1007/978-1-60761-444-9_8 (2010).

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