A New Sequencing Method Identifies Short Peptides with Greater Accuracy

The human body is full of peptides—small chains of amino acids that act as biological messengers, influencing processes ranging from sensory perception to physiological functions. Recent advances in omics technologies have revealed these molecules as key players across a wide range of biological functions, but accurately sequencing short peptides has remained a persistent challenge. Their small size provides limited analytical information, and most conventional identification methods rely on matching data against known protein sequence databases, making it difficult to identify novel peptides not already cataloged.

A study published in Analytical Chemistry from Mitsuru Tanaka and his team at Kyushu University's Faculty of Agriculture describes a method designed to address this gap. The approach uses mass spectrometry combined with de novo sequencing, which makes it possible to identify novel sequences that conventional methods would miss.

The key innovation is the attachment of a coumarin-derived tag to the N-terminus, one end of peptides. The researchers used N-succinimidyl 7-methoxycoumarin-3-carboxylate (Me-Cou) as the tagging reagent. This tag causes the peptide to break apart in a more predictable, stepwise fashion during mass spectrometry analysis, generating clearer fragmentation patterns that can be read like a ladder to determine each peptide's sequence. "Using our approach, the amino acid sequence of peptides can be determined step by step, starting from the tagged end, enabling highly accurate characterization of even the short ones," explains Tanaka.

To test the method, the team analyzed 132 standard peptides with known sequences. Conventional methods correctly identified only 42 of 86 dipeptides and 25 of 46 oligopeptides, producing 32 misidentifications. The new method identified all 132 peptides with zero errors.

The team also applied the method to casein peptone, a complex peptide mixture derived from milk proteins, as a model for evaluating performance on real-world samples. Compared with conventional methods, the approach significantly increased the number and diversity of identified sequences, particularly short peptides consisting of two to ten amino acids. 

"These results demonstrate the accuracy of our approach and its potential for analyzing complex, real-world samples such as fermented foods like sake and soy sauce, as well as biological samples including blood and urine," remarks Tanaka. By enabling more comprehensive characterization of short peptides in biological samples, the method could support future advances in biochemistry and biomedical research.