Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. However, curation and stringent validation further reduces this to 10 reliable identifications. The fragmentation of these 10 peptides was further validated by comparison with the fragmentation of synthetic peptides.

Two peptides (ADDAGAAGGPGGPGGPEMGNRGGFRGGF and MDGEEKTCGGCEGPDAMYVKLISSDGHEFIVKR) were made in-house, while all other peptides were obtained from Thermo Scientific (standard peptide custom synthesis service). In-house peptide synthesis was done using Fmoc-chemistry on an Applied Biosystems 433A Peptide Synthesizer. All required modifications, besides heavy acetylation of primary amines, were introduced during peptide synthesis. Primary amines were blocked after peptide synthesis by adding a 150 times molar excess of an NHS-ester of 13C1D3-acetate, and peptides were incubated for 1 h at 37 °C. This step was repeated once, after which the remainder of the NHS-ester was quenched by adding glycine to a final concentration of 30 mM and incubating the peptides for 10 min at room temperature. O-acetylation was reversed by adding hydroxylamine (75 mM f.c.) followed by an incubation for 10 min at room temperature. Next, peptides were purified on OMIX C18 Tips (Agilent) which were first washed with pre-wash buffer (0.1% TFA in water/acetonitrile (20:80, v/v) and pre-equilibrated with 0.1% TFA before sample loading. Tips were then washed with 0.1% TFA and peptides were eluted with 0.1% TFA in water/acetonitrile (40:60, v/v). Purified peptides were mixed and diluted to a final concentration of 100 fmol/µl (of each peptide). 1 pmol of the acetylated synthetic peptides was injected for LC-MS/MS analysis on an Ultimate 3000 RSLCnano system in-line connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo). Trapping was performed at 10 μl/min for 4 min in loading solvent A on a 20 mm trapping column (made in-house, 100 μm internal diameter (I.D.), 5 μm beads, C18 Reprosil-HD, Dr. Maisch, Germany). The peptides were separated on a 200 cm µPAC™ column (C18-endcapped functionality, 300 µm wide channels, 5 µm porous-shell pillars, inter pillar distance of 2.5 µm and a depth of 20 µm; Pharmafluidics, Belgium). The column was kept at a constant temperature of 50 °C. Peptides were eluted by a linear gradient reaching 26.4% MS solvent B after 20 min, 44% MS solvent B after 25 min and 56% MS solvent B at 28 min, followed by a 5-minutes wash at 56% MS solvent B and re-equilibration with MS solvent A. The first 15 min, the flow rate was set to 750 nl/min after which it was kept constant at 300 nl/min. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition with the m/z-values of the precursors of the synthetic peptides as an inclusion list. Full-scan MS spectra (300-1500 m/z) were acquired in 3 s acquisition cycles at a resolution of 120,000 in the Orbitrap analyzer after accumulation to a target AGC value of 200,000 with a maximum injection time of 30 ms. The precursor ions not present in the inclusion list were filtered for charge states (2-7 required) and intensity (minimal intensity of 5E3). The precursor ions were selected in the ion routing multipole with an isolation window of 1.6 Da and accumulated to an AGC target of 10E3 or a maximum injection time of 40 ms and activated using CID fragmentation (35% NCE). The fragments were analyzed in the Ion Trap Analyzer at rapid scan rate. The data analysis software Skyline ((52), Skyline-Daily V21.1.1.316, was used to compare the ranking of the fragment ions between the synthetic peptides and the possible NTR peptides (identified by a previous experiment). For each synthetic peptide, the top 10 most abundant fragment ions of the synthetic peptides were selected to perform the comparison. A previously identified N-terminal peptide was considered to matching a synthetic peptide if the ranking of the fragment ions was in line with the ranking of the fragment ions of the synthetic peptide.

Two peptides (ADDAGAAGGPGGPGGPEMGNRGGFRGGF and MDGEEKTCGGCEGPDAMYVKLISSDGHEFIVKR) were made in-house, while all other peptides were obtained from Thermo Scientific (standard peptide custom synthesis service). In-house peptide synthesis was done using Fmoc-chemistry on an Applied Biosystems 433A Peptide Synthesizer. All required modifications, besides heavy acetylation of primary amines, were introduced during peptide synthesis. Primary amines were blocked after peptide synthesis by adding a 150 times molar excess of an NHS-ester of 13C1D3-acetate, and peptides were incubated for 1 h at 37 °C. This step was repeated once, after which the remainder of the NHS-ester was quenched by adding glycine to a final concentration of 30 mM and incubating the peptides for 10 min at room temperature. O-acetylation was reversed by adding hydroxylamine (75 mM f.c.) followed by an incubation for 10 min at room temperature. Next, peptides were purified on OMIX C18 Tips (Agilent) which were first washed with pre-wash buffer (0.1% TFA in water/acetonitrile (20:80, v/v) and pre-equilibrated with 0.1% TFA before sample loading. Tips were then washed with 0.1% TFA and peptides were eluted with 0.1% TFA in water/acetonitrile (40:60, v/v). Purified peptides were mixed and diluted to a final concentration of 100 fmol/µl (of each peptide).