Table 1.
Summary of the techniques discussed in this review.
| Technique | Overview of rationale | What is measured | Selected references |
|---|---|---|---|
| Radiolabeling | Newly synthesized proteins incorporate radiolabeled amino acids. | Radioactive emission from total protein reflects the translation status of the sample. | Lageix et al., 2008; Galland et al., 2014; Wang et al., 2017 |
| FUNCAT | Newly synthesized proteins incorporate a non-canonical amino acid that can be detected by “click” chemistry. | Fluorescence from a “click” chemistry reaction reflects the translation status of the sample. | Tom Dieck et al., 2012; Glenn et al., 2017 |
| Cell-free protein expression system | The conditions for translation can be tightly controlled and exogenous elements easily introduced in a cell-free system. | The abundance of the protein of interest is examined under customizable conditions. | Murota et al., 2011; Buntru et al., 2014; Alvarez et al., 2016 |
| Translational reporter fusion | A reporter gene that is easily detected at the protein level by fluorescence, antibodies, etc., is fused to a gene of interest. | The abundance of a reporter protein is detected as a surrogate for the protein of interest. | Tabuchi et al., 2006; Eastmond et al., 2010; Paik et al., 2012 |
| SILAC | A pulse of isotope-labeled amino acids specifically marks newly synthesized proteins. | The presence of an isotope distinguishes new proteins upon MS analysis. | Ong et al., 2002; Gruhler et al., 2005; Schwanhausser et al., 2009; Lewandowska et al., 2013 |
| BONCAT | Newly synthesized proteins incorporate a non-canonical amino acid that can be isolated by “click” chemistry. | MS analysis identifies affinity-purified proteins. | Glenn et al., 2017 |
| QuanCAT | A pulse of non-canonical amino acids specifically marks newly synthesized proteins that can be isolated by “click” chemistry. | MS analysis identifies affinity-purified, newly synthesized proteins. | Howden et al., 2013 |
| Puromycylation | Puromycin “tags” newly synthesized proteins. | Puromycin incorporation serves as a surrogate for global translation. | Basbouss-Serhal et al., 2015 |
| SunSeT | Newly synthesized proteins incorporate puromycin, which can be detected with antibodies. | Puromycin incorporation serves as a surrogate for translation. | Schmidt et al., 2009; Van Hoewyk, 2016 |
| RPM | Puromycin “tags” newly synthesized proteins and a chemical locks ribosomes in place. | Puromycin detection reveals the location of nascent peptides. | Seedhom et al., 2016 |
| PUNCH-P | Biotinylated puromycin “tags” nascent peptides for isolation with streptavidin beads. | Purified proteins are analyzed by MS. | Aviner et al., 2013 |
| Polysome profiling | Ribosome:mRNA complexes are separated using ultracentrifugation through a sucrose gradient. | The distribution of ribosomes shows global translation trends; the abundance of transcripts in different fractions is detected by qPCR, sequencing, etc. | Kawaguchi et al., 2004; Mustroph et al., 2009a; Karginov and Hannon, 2013; Yanguez et al., 2013; Layat et al., 2014; Basbouss-Serhal et al., 2015; Bai et al., 2017 |
| RDM | The number of ribosomes on a transcript alters the sedimentation rate of the transcript. | Ribosome density is deduced from the size and fractionation distribution of mRNA fragments. | Arava et al., 2005 |
| Toeprinting | A ribosome present on a transcript blocks RT and produces truncated cDNA products. | The length of RT products indicates the presence or absence of ribosomes on a transcript. | Anthony and Merrick, 1992; Gould et al., 2005; Hayashi et al., 2017 |
| Ribosome footprinting (Ribo-seq) | A ribosome present on a transcript protects the transcript from RNase digestion, leaving behind ribosome “footprints”. | Ribosome footprints are sequenced to reveal the location and average density of ribosomes across the transcriptome. | Liu et al., 2013; Juntawong et al., 2014; Lei et al., 2015; Li et al., 2015; Merchante et al., 2015; Chotewutmontri and Barkan, 2016; Hsu et al., 2016; Lukoszek et al., 2016; Bazin et al., 2017; Xu et al., 2017; Zoschke et al., 2017 |
| TCP-seq | Crosslinking of ribosomes to transcripts allows detection of ribosome subunit:mRNA interactions. | Ribosome footprints from small subunit and monosome fractions are sequenced to reveal translation dynamics. | Archer et al., 2016 |
| TRAP, TRAP-seq | Tagged ribosomes with associated transcripts are affinity purified from cellular lysate. | The sequence of ribosome-bound transcripts identifies the translatome. | Zanetti et al., 2005; Mustroph et al., 2009b; Jiao and Meyerowitz, 2010; Rajasundaram et al., 2014; Wang and Jiao, 2014; Vragovic et al., 2015 |
| Gaussia luciferase | “Flash” kinetics allow for live visualization of newly synthesized protein of interest. | Bioluminescence acts as a marker of tagged, newly synthesized proteins. | Na et al., 2016 |
| Photoswitchable protein | Exposure to UV light changes the emission spectrum of tagged protein of interest. | Fluorescent emission in the original spectrum acts as a surrogate for newly synthesized proteins. | Leung et al., 2006; Raab-Graham et al., 2006; Leung and Holt, 2008; Vogelaar et al., 2009 |
| Ribosome “knock-off” | An advancing ribosome knocks the fluorescent, hairpin-specific RNA binding protein off of the transcript of interest. | A change is fluorescent signal localization acts as an indicator of active translation. | Halstead et al., 2015 |
| Fluorescent colocalization | A fluorescently marked transcript co-localizes with a fluorescently marked ribosome or nascent peptide. | A change in fluorescent signal localization or new fluorescent foci acts as an indicator of active translation. | Katz et al., 2016; Morisaki et al., 2016; Wang et al., 2016; Wu et al., 2016; Yan et al., 2016 |
| Splinted PCR | A “splint” oligonucleotide facilitates the transcript-specific ligation of an adapter to decapped transcripts. | An RT-PCR or qRT-PCR product is detected as evidence of decapped/degraded transcripts. | Hu et al., 2009; Blewett et al., 2011; Merret et al., 2015 |
| PARE, GMUCT | Decapped or cleaved transcripts have a free 5′-monophosphate that can be directly ligated for library preparation. | Sequenced transcripts reveal the degradome; periodicity can indicate co-translational decay. | German et al., 2008; Gregory et al., 2008; Willmann et al., 2014; Hou et al., 2016; Yu et al., 2016; Crisp et al., 2017 |
| 5PSeq | Decapped or cleaved transcripts have a free 5′-monophosphate that is directly ligated for library preparation. | Sequenced transcripts from capped and uncapped fractions reveal the degradome; periodicity can indicate co-translational decay. | Pelechano et al., 2015; Pelechano and Alepuz, 2017 |