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. 2019 Jul 18;20(14):3524. doi: 10.3390/ijms20143524

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.