Table 1.
Methodology | Incubation Time | Considerations | Current Model Utility | Refs. | |
---|---|---|---|---|---|
pSILAC: Pulsed Stable Isotopic Labeling of Amino acids | Pulsed isotopic labeling of amino acids | Short to Long | Robust incorporation but generally requires long incubation times. May introduce a bias of tag incorporation. | In vitro | [31,32] |
BONCAT: Bio-orthogonal Non-Canonical Amino acid Tagging | Non-canonical amino acid incorporation and chemical capture | Short to Medium | Weak incorporation at shorter incubation timescales. Can be adapted for fluorescent detection. Strong MS detection after purification. | In vitro Ex vivo In vivo |
[35,36,37,38,39,40,41,42,43,44,45,46,47,49,50] |
PALM: Pulsed Azidohomoalanine Labeling in Mammals | In vivo BONCAT using AHA-enriched feed | Long | Weak incorporation and requires multi-day diet on enriched feed. Nascent translation can be detected in sub-cellular fractions. | In vivo | [48] |
BONLAC: Combinatorial BONCAT and pSILAC | Combined pSILAC with BONCAT enrichment | Medium | Enables the robust detection of nascent peptides but with a greater experimental complexity. | In vitro Ex vivo |
[41,52,53] |
mMetRS BONCAT: Mutated Methionyl-tRNA synthetase coupled with BONCAT | BONCAT but with cell-specific expression of expanded tRNAs | Medium | Requires genetic manipulation or viral-mediated genetic transfer but can be adapted for cell-specific investigations of nascent translation. | In vitro In vivo |
[59,60,61,62] |
Puromycin | Puromycin labeling and affinity capture | Short | Requires simple injection followed by affinity capture. Can inhibit translation at high concentrations. | In vitro Ex vivo In vivo |
[51,65,67,68] |
PUNCH-P: Puromycin associated Nascent Chain Proteomics | Puromycin-biotin labeling and chemical capture | Short | Requires tissue homogenization prior to incubation but with strong incorporation. | In vitro In vivo& |
[70,72] |
Experimental incubation times range can be short (five minutes to one hour), medium (over one hour to six hours), or long (over six hours). The current model utility describes the sample conditions used in nascent proteomic techniques discussed in this review. Techniques with in vivo utility denote previously published studies where the chosen tag was incorporated into nascent proteins in live animals for downstream analysis. & PUNCH-P requires tissue homogenization prior to puromycin-biotin incorporation for in vivo use.