Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

mESC Cell Culture and Expansion in Feeder Free Medium

E14Tg2a mESC cells (a kind gift of Prof. I. Chambers, University of Edinburgh, Scotland, UK) were maintained at 21% O₂, 5% CO₂ and passaged every 2 days on 0.1% gelatin (Chemicon)-coated dishes using TripLE^TM Express (Invitrogen) as a cell-dissociating agent in 80% KO DMEM high glucose, 20% KO serum replacement, 4 mm l-glutamine, 0.1 mm penicillin/streptomycin, 1× nonessential amino acids, 1 mm sodium pyruvate, 110 mm 2-mercaptoethanol (all from Invitrogen), supplemented with 1 unit/ml murine leukaemia inhibitory factor (Chemicon), 1 mm MEK inhibitor PD0325901 (Axon MedChem), and 3 mm GSK3 inhibitor CHIR99021 (Axon MedChem) (23).

Shotgun Proteome Analysis

15 × 10⁶ mESC E14 cells were lysed by three rounds of freeze-thaw lysis in 1 ml of 50 mm NH₄HCO₃ (pH 7.9). Lysates were cleared by centrifugation for 15 min at 16,000 × g. Protein concentrations were measured using the protein assay kit (Bio-Rad) according to the manufacturer's instructions. To partially denature proteins, guanidinium hydrochloride (final concentration 0.5 m) and acetonitrile (final concentration 2%) were added to the cleared protein extracts. 1 mg of the protein sample was digested overnight at 37°C using sequencing-grade modified trypsin (Promega, Madison, WI) (enzyme/substrate of 1:100 w/w). Samples were acidified with acidic acid to a final concentration of 0.5%. The digest was vacuum-dried, and the equivalent of 250 μg of the original protein material was loaded onto a reverse phase-HPLC column for fractionation as described previously (24). To prevent oxidation of methionines between reverse phase-HPLC runs, methionines were oxidized in the injector compartment by transferring 20 μl of a freshly prepared aqueous 3% H₂O₂ solution to a vial containing 90 μl of the acidified peptide mixture (final concentration of 0.54% H₂O₂). This reaction proceeded for 30 min at 30°C after which the sample was immediately injected onto the reverse phase-HPLC column and separated. Fractions of 0.5 min were collected from 20 to 80 min after sample injection (120 fractions). These peptide fractions were vacuum-dried, and fractions eluting 12 min apart were pooled by re-dissolving these in a final volume of 40 μl of 2 mm tris(2-carboxyethyl)phosphine and 2% acetonitrile, similar to a pooling strategy described previously (24). In total, 24 samples were analyzed by LC-MS/MS.

N-terminal COFRADIC Analysis

Cell lysis, N-terminal COFRADIC analyses, and sample analyses were performed as described previously (25). To enable the assignment of in vivo N-terminal acetylation events, all primary protein amines were blocked making use of an (stable isotopically encoded) N-hydroxysuccinimide ester at the protein level (i.e. N-hydroxysuccinimide-[¹³C₂D₃]acetate). In total, 45 samples were analyzed by LC-MS/MS.

LC-MS/MS Analysis Using an Ultimate 3000 RSLC Nano-LTQ Orbitrap Velos System

LC-MS/MS analysis was performed using an Ultimate 3000 RSLC nano-LC-MS/MS system (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany). 2 μl of the sample mixture was first loaded on a trapping column (made in-house, 100 μm internal diameter × 20 mm long, 5-μm Reprosil-Pur Basic-C18-HD beads, Dr. Maisch, Ammerbuch-Entringen, Germany). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 μm internal diameter × 150 mm long, 3-μm C18 Reprosil-Pur Basic-C18-HD beads). Peptides were loaded with solvent A′ (0.1% trifluoroacetic acid in 2% acetonitrile) and were separated with a linear gradient from 98% of solvent A″ (0.1% formic acid in 2% acetonitrile) to 50% of solvent B′ (0.1% formic acid in 80% acetonitrile) with a linear gradient of 1.8% of solvent B′ increase per min at a flow rate of 300 nl/min, followed by a steep increase to 100% of solvent B′. The Orbitrap Velos mass spectrometer was operated in a data-dependent mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in an MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The 10 most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 20 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.2.0, Matrix Science). While generating these peak lists, a grouping of spectra was allowed with a maximum intermediate retention time of 30 s and maximum intermediate scan count of 5. Grouping was done with a 0.005-Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping, and the relative signal-to-noise limit was set at 2.

Peptide/Protein Identification and Interpretation

The obtained fragmentation spectra were searched against the custom database (combination of UniProtKB-SwissProt and RIBO-seq-derived translation sequences) using three different search engines as follows: OMSSA (version 2.1.9), X!Tandem (TORNADO, version 2010.01.01.04) and Mascot (version 2.3). The first two were run from the SearchGUI graphical user interface, version 1.10.4 (26). A combination of X!Tandem and Mascot was used for the N-terminal COFRADIC analysis, a combination of all three search engines for the shotgun proteome analysis. Note that OMMSA cannot cope with the protease setting semi-ArgC/P needed to analyze the N-terminal COFRADIC data (see below).

For the shotgun proteome data, trypsin was set as a cleavage enzyme allowing for one missed cleavage, and singly to triply charged precursors or singly to quadruply charged precursors were taken into account, respectively, for the Mascot or X!Tandem/OMSSA search engines, and the precursor and fragment mass tolerance were set to 10 ppm and 0.5 Da, respectively. Methionine oxidation to methionine sulfoxide, pyroglutamate formation of N-terminal glutamine, and acetylation (protein N terminus) were set as variable modifications. For the N-terminal COFRADIC analysis, the protease setting semi-ArgC/P (Arg-C specificity with arginine-proline cleavage allowed) was used. No missed cleavages were allowed, and the precursor and fragment mass tolerance were also set to 10 ppm and 0.5 Da, respectively. Carbamidomethylation of cysteine and methionine oxidation to methionine sulfoxide and ¹³C₃D₂ acetylation of lysines were set as fixed modifications. Peptide N-terminal acetylation or ¹³C₃D₂ acetylation and pyroglutamate formation of N-terminal glutamine were set as variable modifications, and instrument setting was put on ESI-TRAP.

Protein and peptide identifications in addition to data interpretations were done using the PeptideShaker algorithm, setting the false discovery rate to 1% at all levels (protein, peptide, and peptide to spectrum matching). Aforementioned tools and algorithms (SearchGui, X!Tandem, OMSSA, and PeptideShaker) are freely available as open source.

Ribosome Profiling

Raw sequencing reads of the mESC ribosome profiling data (15) were downloaded from the Gene Expression Omnibus (dataset GSE30839). All reads from the control (cycloheximide-treated sample GSM765292) and harringtonine-treated (sample GSM765295) were remapped using bowtie (version 0.12.7) on the mouse genome (assembly version 37) using the protocol described (16).

Genome-wide Visualization

Genome-wide visualization of the experimental data, in combination with publicly available data, was accomplished using an in-house-developed genome browser (H2G2). Information tracks containing the ribosome profile mappings of the cycloheximide-treated sample (generating a translation profile all over the coding mRNA) and harringtonine-treated sample (translation profile accumulation at the TIS) are available (see Ref. 15 for more information). Furthermore, an information track was constructed showing the predicted translation products track, based on the TIS predictions from Ingolia et al. (15) and the UCSC transcript annotation. The genomic locations of the N-terminal peptides identified with the COFRADIC experiment are also visualized in the H2G2 browser. In addition, several other information tracks are available for visualizing public data as follows: genomic information from a local Ensembl (7) instance (NCBIM37.66) and PhastCons conservation scores (27), among others. More information on how to use the H2G2 browser and what login credentials to use can be found in the supplemental File S1.

Swiss-Prot/RIBO-seq Integrated Database Construction

The combined protein database was constructed using translation products derived from the mouse RIBO-seq data presented by Ingolia et al. (15) and all mouse UniProtKB-SwissProt (6) protein sequences (downloaded version 2012_10). Fig. 1 gives an overall representation of the identification strategy. The RIBO-seq-derived translation products were reconstructed based on both the predicted (a)TIS genomic locations (15) and the corresponding mRNA sequences obtained from the UCSC Genome Browser resource (table browser: assembly = NCBI37/mm9, track = Old UCSC Genes). After reconstructing the amino acid sequences, the UCSC identifiers were mapped to UniProtKB-SwissProt identifiers (to safeguard uniformity) using the UCSC gene annotation information (mm9.knownGene table using the Table browser). For several UCSC IDs, mapping to a RefSeq and thus not to a UniProtKB-SwissProt identifier, the Uniprot Mapping Service was applied to retrieve the corresponding UniProtKB-TrEMBL identifiers. Finally, to remove redundancy, introduced by the combination of the RIBO-seq-derived translation products and the set of known UniProtKB-SwissProt protein sequences, duplicated sequences were removed, retaining the UniProtKB-SwissProt sequence where possible. Moreover, only the longest form of a series of gene translation products (N-terminal extended or canonical) is withheld in the combined database. The custom database contains 24,355 sequences as compared with UniProtKB-SwissProt version 2012_10 holding 16,570 proteins. We opted to merge the RIBO-seq-derived translation products to the UniProtKB-SwissProt protein repository for several reasons as follows: (i) Swiss-Prot contains only reviewed, maintained, and well annotated sequences and thus serves well as a species-specific base-line protein repository; (ii) opting for Swiss-Prot instead of trEMBL (containing the UniProtKB unreviewed entries and hence also partial sequences) makes the downstream protein inference task less complex, and (iii) sample specific translation products that are missing in the Swiss-Prot reference will be added to the custom database because they are contained in the RIBO-seq-derived translation products sequence set.

Shotgun Proteomics

Using the custom combined database as search space, the number of protein identifications increases with 2.64% (from 3,166 to 3,252 protein identifications) as compared with searching the UniProtKB-SwissProt reference set only (see Fig. 2). Along the same line, the number of peptide identifications, using the same 1% false discovery rate threshold, increases with 4.93% (from 29,343 to 30,865 peptide identifications). The majority of these newly identified proteins are recorded in UniProtKB/trEMBL. Only a few originate from peptide identifications that overlap (part of) an N-terminal extension, an exonic region of an alternative spliced isoform, or an SNP mutation site. Similarly and due to the increased protein coverage, these phenomena account for the improved protein identification and score significance for another 1.5% of proteins. Twenty seven protein identifications have an increased coverage because peptide(s) were identified that coincide with a mutation site, another 11 proteins because peptide(s) are partly contained in the N-terminal extension of the RIBO-seq-derived translation product, and another 11 proteins because peptides are located within an exonic region of an alternative translation product (see Fig. 3 exemplifying these three categories). All protein identifications, grouped into the different categories (Fig. 2), are provided as supplemental Table S1. Also, the different search engine performances, measured as validated peptide-to-spectrum matches, are listed in supplemental Table S2.

N-terminal COFRADIC

Protein inference is responsible for the fact that specific ORFs (i.e. truncated protein forms) are difficult to monitor using conventional LC-MS/MS-based analyses of total protein digests. Because the protein N termini are highly indicative for the N-terminal protein isoforms expressed and thus indicative for the TIS identified by means of GTI-seq, we applied the N-terminal COFRADIC positional proteomics approach, strongly enriching for protein N-terminal peptides (28). Prior to tryptic digestion, all primary amines are modified by ¹³C₂D₃ acetylation, which allows us to differentiate between in vivo-acetylated and free N termini (in vitro ¹³C₂D₃-acetylated) by introducing a spacing of 5 Da between these types of N-terminal peptides. If needed, this labeling strategy allows for a straightforward calculation of the extent of N-terminal acetylation (29). After tryptic digestion, all protein N-terminal peptides will thus be blocked, whereas internal peptides acquired a newly generated primary α-amine, a property exploited to isolate N-terminal peptides from internal peptides in a diagonal chromatography setup that follows strong cation exchange enrichment at low pH (25).

To select for TIS-indicative N termini, we relied on the co-translational nature of N-terminal acetylation of protein N termini (30) by N-terminal acetyltransferases, the near universal requirement of a Met-encoding (or near-cognate) initiator codon (iMet) and the co-translational processing of iMet by methionine aminopeptidases (rules reported in Ref. 31). Following LC-MS/MS analyses and combined database searching, we identified 1,835 TIS-indicative N termini; an overview of the different categories of these N termini is presented in Fig. 4. The majority of the identified N termini map to canonical translation start sites (1,556 peptides, 84.8%). Another 259 peptides (14.1%) start beyond protein position 2 and are indicative for alternative or wrongly annotated protein translation initiation sites. The peptides positioned in RIBO-seq-annotated N-terminal extensions (16 occurrences, 0.9%) and uORFs (completely within the 5′UTR or out-of-frame and overlapping with canonical CDS; 4 occurrences, 0.2%) could only be identified using the custom combined database strategy. These extended translation products point to upstream TIS (uTIS). Compared with the RIBO-seq sequencing results (see supplemental Fig. S1) (15), it is clear that the N-terminal COFRADIC technique only validated a small number of translated uORFs and N-terminal extensions (relative to the annotated CDS), although in sharp contrast, nearly half of the identified translation products from the RIBO-seq results consist of translated uORFs (supplemental Fig. S1). Although N-terminal COFRADIC appears to be more sensitive as compared with a regular shotgun proteomics for identifying some uORF translation products, it is clear that RIBO-seq outranks both proteomics technologies.

A listing of all identified N-terminal peptides, categorized into the different classes (canonical, extension, truncation, uORF, and overlapping uORF) is provided as supplemental Table S3. The different search engine performances, measured as validated peptide-to-spectrum matches, can be found in supplemental Table S2. Furthermore, the N-terminal peptides have also been made available as s visualization track in our in-house developed genome-browser. Next to Ensembl Gene and Transcript visualization tracks (Ensembl annotation version 66 (7)), experimental RIBO-seq data (15) are also presented as custom tracks, allowing manual inspection of the co-occurrence of N-terminal COFRADIC and RIBO-seq experimental evidence. The N-terminal COFRADIC information is also provided in BED-format (32) for upload in a genome browser of choice (supplemental File S2).

Next to annotation of nonregular translation products as (i) N-terminal protein extensions or truncations pointing to aTIS, uTIS, or downstream TIS, or (ii) translation of uORFs, the N-terminal COFRADIC technology provides us with evidence of translation initiation at near-cognate start sites (noninitiator methionine). This was also a remarkable observation within the RIBO-seq studies (13, 15, 18, 19), and notably, we here observe the outcome of this phenomenon at the protein level instead of the mRNA level. Weblogos (33) were created based on the sequence contexts flanking the newly identified uTIS (from the extended protein products) and the TIS of the translated uORFs (see Fig. 6). The weblogos clearly show the presence of translation initiation at near-cognate start codons. Next to the four N termini indicative for N-terminal protein extension starting at the canonical AUG start codon, others were formed by translation initiation at CUG (five N termini), GUG (four N termini), ACG (two N termini), and UUG (one N terminus) (also see supplemental Table S3). These near-cognate start codons respectively encode for leucine, valine, threonine, and again leucine but are recoded to the regular methionine as, for example, exemplified by the N-terminally acetylated iMet-retaining N-terminal MDPPTSEKAVAQGAGR originating from translation initiation of the thyroid receptor-interacting protein 13 transcript at an upstream near-cognate GUG codon. In parallel, next to the newly translated uORF identification starting from the canonical AUG (one N terminus), others were also found starting from near-cognate codons GUG (two N termini), ACG (one N terminus), and UUG (one N terminus) (see supplemental Table S3). In line with these results, the majority of discovered uORFs in RIBO-seq studies was found to start from near-cognate start sites (15, 18, 19). The recoding of the alternative amino acids, resulting from the near-cognate start sites, seems to take place in all identified candidates, pointing to the fact that no non-iMet translation products are formed.

It is also apparent from the sequence context of the start sites that the most crucial Kozak elements (34, 35) are present as follows: a purine at position −3 and a guanine at position +4 within the Kozak motif (A/G)ccAUGG, further corroborating the upstream translation initiation and/or translation of the uORFs.