Table 1 Advantages and disadvantages of micropeptide identification techniques

Ribosome sequencing

Accurate identification of translation start sites

False positives can be produced due to nonspecific binding

Enables protein synthesis processes monitoring in vivo

rRNA and tRNA produce Ribo-Seq noise

39,40

Useful to investigate extension speed, cotranslation processing, etc

Since RNase digests rRNA, it can damage ribosome integrity and cause experimental bias

Mass spectrometry

Direct verification of the protein-coding potential of transcripts

Small protein can be lost during sample preparation and purification

Depends on data from protein databases

45,46

Bioinformatics

A wide range of emerging tools enables, choices to best meet the need

Micropeptides are easy to miss due to their small size

Experiments are needed to confirm the translatability of the identified sORFs

51,52

Other experimental verification

Direct verification of micropeptides

The experimental process is tedious

Determination of the subcellular location of micropeptides and micropeptide interactions with other macromolecules

6,53,57