Identification Of Small Open Reading Frame Encoded Peptides In Human Hepatoma Hep3B Cell Line

Small open reading frame encoded peptide（SEP）is one kind of peptide with sequence length less than 100AAs,and it is encoded by small open reading frame（sORF）.SEPs have essential functions and can be found in many species.Bioinformatic analysis suggested there are still a large amount of predicted SEPs,which need to be discovered and characterized.SEPs identification is the first and critical step for their function researches.Thus,high efficiency identification of SEPs will be greatly significant.Mass spectrometry（MS）based proteomics assay can detect the peptides translated from these ORFs and confirm the translation.However,the identification efficiency is quite low due to the properties of SEPs,low molecular and low abundance of SEPs.In order to improve the identification efficiency,we use organic solvent precipitation method from traditional peptidomics for SEPs extraction and enrichment,and combine two different separation methods,which is Tricine-SDS-PAGE and electrostatic repulsion-hydrophilic interaction chromatography（ERLIC）fraction.We identified the SEPs in the human hepatoma Hep3B cell line.First,we compare the efficiency of different methods in Microproteins and SEPs enrichment and identification.We found that ACN extraction is more effective in lower molecular and hydrophobic microproteins and SEPs enrichment,and result in higher intensity and cleaner spectrum background in following LC-MS/MS detection.Although HCl extraction leads to interfering peaks in the spectrum background,it can achieve higher sequence coverage.Appropriate separation methods after enrichment can improve the quantity and quality of identified microproteins and SEPs,indicating the combination of different methods is complementary and could improve the efficiency of SEPs identification.Subsequently,we screened the identified microproteins and SEPs by removing the peptides in annotated proteins and completely homology to known proteins.That gave us 271 novel SEPs with 285 unique peptides in total.Among them,3 SEPs have translation evidence,while others are just predicted in previous researches.From the transcription and gene location information,we can find that 85%in the 271 new SEPs were encoded by the traditional "non-coding" region.Finally,we searched domains and homology of these new SEPs in our data,70 SEPs have predicted domains and 114 SEPs with homologies across species,which indicated they may play functional role in biological processes.For example,IP₆61898 have COX6C domain,it is homologous to Cytochrome c oxidase subunit 6C with 76.1%identity and conserves across seven species.IP₇89354 have TCTP domain,it is homologous to translationally-controlled tumor protein（TPT1）with 80%identity and conserves across six species.At the same time,we identify 22 uORF-encoded peptides,which may play a role in translational control of their downstream genes.In this paper,we improved the SEPs identification efficiency by combining different extraction and separation methods.Meanwhile,we discover and characterize SEPs in Hep3B cell line,which providing robust evidence for the coding potential of these small open reading frames.Results in this work are the expansion of the human genome and proteome,and may provide theoretical and data support for the further function research of these SEPs.