Comparative Proteomic Profiling of Unannotated Microproteins and Alternative Proteins in Human Cell Lines

Abstract

Ribosome profiling and mass spectrometry have revealed thousands of small and alternative open reading frames (sm/alt-ORFs) that are translated into polypeptides variously termed as microproteins and alt-proteins in mammalian cells. Some micro-/alt-proteins exhibit stress-, cell-type-, and/or tissue-specific expression; understanding this regulated expression will be critical to elucidating their functions. While differential translation has been inferred by ribosome profiling, quantitative mass spectrometry-based proteomics is needed for direct comparison of microprotein and alt-protein expression between samples and conditions. However, while label-free quantitative proteomics has been applied to detect stress-dependent expression of bacterial microproteins, this approach has not yet been demonstrated for analysis of differential expression of unannotated ORFs in the more complex human proteome. Here, we present global micro-/alt-protein quantitation in two human leukemia cell lines, K562 and MOLT4. We identify 12 unannotated proteins that are differentially expressed in these cell lines. The expression of six micro/alt-proteins from cDNA was validated biochemically, and two were found to localize to the nucleus. Thus, we demonstrate that label-free comparative proteomics enables quantitation of micro-/alt-protein expression between human cell lines. We anticipate that this workflow will enable the discovery of regulated sm/alt-ORF products across many biological conditions in human cells.

Complementarity of Different SDS‐PAGE Gel Staining Methods for the Identification of Short Open Reading Frame‐Encoded Peptides

“…[28] Recently, Cao et al suggested a peptide-centric approach [31] to validate SEP identified with only one PSM. [17] We performed this additional filtering step and in total 39 out of 45 SEP identified in or study passed this criterion, while six peptides assigned to SEP identified with only one PSM were negative (Table 1). We further manually interpreted the six spectra of the peptides not passed the criterion and observed extensive precursor co-isolation of other peptides.…”

“…Here, we investigated classical SDS-PAGE based proteome separation followed by in-gel digestion with trypsin prior to LC-MS/MS (GeLC-MS) analysis for the identification of small proteins, and in particular SEP. [16,17] We assessed the influence of different SDS-PAGE gel staining methods prior to in-gel digestion: i) only fixing the gel, ii) zinc-imidazole negative staining, [18] and iii) Coomassie-staining, on the identification of SEP. As a model organism, we used the archaeon Methanosarcina mazei, grown under nitrogen starvation conditions; the same biological situation was used earlier to establish alternative strategies for SEP analysis. [11,14] The genome of this organism encodes for ≈3400 predicted proteins and additional about 1400 predicted SEP. [19][20][21]…”

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24

“… a Control HEK 293T cells (lane 1) or alt-RPL36-GFP11-FLAG knock-in (KI) HEK 293T cells (lane 2) were transfected with alt-LEF1-FLAG (which is a recently identified ~20 kDa alt-ORF[ 1 ]), serving as a FLAG-IP control, and IPs were performed with anti-FLAG antibody followed by IB with anti-FLAG. Cell lysates (1%) before IP (input) were used as the loading controls.…”

“…Thousands of previously unannotated small and alternative open reading frames (smORFs, <100 amino acids, and alt-ORFs, >100 amino acids, respectively) have recently been revealed via genomic and proteomic technologies in mammalian genomes 1 , 2 . These genes previously escaped annotation not just because of their short length, but because, as a class, they exhibit low homology to proteins of known function, and are enriched for initiation at near-cognate non-AUG start codons (~50%) 3 , 4 .…”

cncRNAdb: a manually curated resource of experimentally supported RNAs with both protein-coding and noncoding function

“…In a sense, the discovery of cncRNA blurred the boundary between ‘coding’ and ‘noncoding’ RNAs and led researchers to reconsider the function, evolution and understanding of RNAs ( 8 , 25 , 37 , 38 ). Recently, an increasing number of cncRNAs have been identified, including translated ncRNAs and untranslated mRNAs ( 18 , 39–41 ), and some studies began to identify cncRNAs by combining different high-throughput experimental technologies, such as mass spectrometry, ribosome profiling and CRISPR-based screening ( 18 , 24 , 42 , 43 ). Here, we developed cncRNAdb ( http://www.rna-society.org/cncrnadb/ ), a manually curated database of experimentally supported cncRNAs, which aims to provide a resource for efficient manipulation, browsing and analysis of cncRNAs.…”