Identification of phosphorylated small ORF-encoded peptides in Hep3B cells by LC/MS/MS.

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO2 materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SIGNIFICANCE: Small ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation.

Identified Small Open Reading Frame-Encoded Peptides in Human Serum with Nanoparticle Protein Coronas.

The low-molecular-weight proteins (LMWP) in serum and plasma are related to various human diseases and can be valuable biomarkers. A small open reading frame-encoded peptide (SEP) is one kind of LMWP, which has been found to function in many bioprocesses and has also been found in human blood, making it a potential biomarker. The detection of LMWP by a mass spectrometry (MS)-based proteomic assay is often inhibited by the wide dynamic range of serum/plasma protein abundance. Nanoparticle protein coronas are a newly emerging protein enrichment method. To analyze SEPs in human serum, we have developed a protocol integrated with nanoparticle protein coronas and liquid chromatography (LC)/MS/MS. With three nanoparticles, TiO2, Fe3O4@SiO2, and Fe3O4@SiO2@TiO2, we identified 164 new SEPs in the human serum sample. Fe3O4@SiO2 and a nanoparticle mixture obtained the maximum number and the largest proportion of identified SEPs, respectively. Compared with acetonitrile-based extraction, nanoparticle protein coronas can cover more small proteins and SEPs. The magnetic nanoparticle is also fit for high-throughput parallel protein separation before LC/MS. This method is fast, efficient, reproducible, and easy to operate in 96-well plates and centrifuge tubes, which will benefit the research on SEPs and biomarkers.

Microprotein Dysregulation in the Serum of Patients with Atrial Fibrillation.

The incidence rate of atrial fibrillation (AF) has stayed at a high level in recent years. Despite the intensive efforts to study the pathologic changes of AF, the molecular mechanism of disease development remains unclarified. Microproteins are ribosomally translated gene products from small open reading frames (sORFs) and are found to play crucial biological functions, while remain rare attention and indistinct in AF study. In this work, we recruited 65 AF patients and 65 healthy subjects for microproteomic profiling. By differential analysis and cross-validation between independent datasets, a total of 4 microproteins were identified as significantly different, including 3 annotated ones and 1 novel one. Additionally, we established a diagnostic model with either microproteins or global proteins by machine learning methods and found the model with microproteins achieved comparable and excellent performance as that with global proteins. Our results confirmed the abnormal expression of microproteins in AF and may provide new perspectives on the mechanism study of AF.

Molecular Rewiring of the Jasmonate Signaling Pathway to Control Auxin-Responsive Gene Expression.

The plant hormone jasmonic acid (JA) has an important role in many aspects of plant defense response and developmental process. JA triggers interaction between the F-box protein COI1 and the transcriptional repressors of the JAZ family that leads the later to proteasomal degradation. The Jas-motif of JAZs is critical for mediating the COI1 and JAZs interaction in the presence of JA. Here, by using the protoplast transient gene expression system we reported that the Jas-motif of JAZ1 was necessary and sufficient to target a foreign reporter protein for COI1-facilitated degradation. We fused the Jas-motif to the SHY2 transcriptional repressor of auxin signaling pathway to create a chimeric protein JaSHY. Interestingly, JaSHY retained the transcriptional repressor function while become degradable by the JA coreceptor COI1 in a JA-dependent fashion. Moreover, the JA-induced and COI1-facilitated degradation of JaSHY led to activation of a synthetic auxin-responsive promoter activity. These results showed that the modular components of JA signal transduction pathway can be artificially redirected to regulate auxin signaling pathway and control auxin-responsive gene expression. Our work provides a general strategy for using synthetic biology approaches to explore and design cell signaling networks to generate new cellular functions in plant systems.