TY - JOUR
T1 - ProTInSeq
T2 - transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs
AU - Miravet-Verde, Samuel
AU - Mazzolini, Rocco
AU - Segura-Morales, Carolina
AU - Broto, Alicia
AU - Lluch-Senar, Maria
AU - Serrano, Luis
N1 - Publisher Copyright:
© The Author(s) 2024.
PY - 2024/3/7
Y1 - 2024/3/7
N2 - Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach.
AB - Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach.
UR - http://www.scopus.com/inward/record.url?scp=85187105749&partnerID=8YFLogxK
U2 - 10.1038/s41467-024-46112-2
DO - 10.1038/s41467-024-46112-2
M3 - Article
C2 - 38453908
AN - SCOPUS:85187105749
SN - 2041-1723
VL - 15
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 2091
ER -