Table 1.

Comparison of proteomic techniques that focus on the nascent proteome and experimental considerations.

	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.