Targeted proteomics informed by long-read RNA-sequencing reveals protein isoform expression associated with bone mineral density
Abstract
The expanding catalog of sQTLs from projects like GTEx has established that genetic risk for complex traits is frequently mediated through RNA splicing. Yet it is generally unknown whether disease-associated splice events produce stable, distinct protein isoforms and whether they are functionally consequent. Even the deepest shotgun proteomics efforts only capture a minority of annotated splice events; isoform-specific peptides are systematically under-sampled by nature of being lower abundance and intrinsically harder to detect. Here, we implement a genetics-guided targeted mass spectrometry (MS) strategy to detect splice-derived protein isoforms prioritized by sQTL colocalization. Using internal standard parallel reaction monitoring (IS-PRM), we targeted 288 peptides corresponding to 166 putative BMD-associated isoforms in differentiating osteoprogenitor cells and patient-derived primary mesenchymal stem cells (MSCs). Targeted acquisition increased recovery of isoform-informative peptides relative to conventional DDA, rescued splice events that were undetected or sparsely sampled under global acquisition, and confirmed protein-level expression of genetically prioritized isoforms in clinical material. Together, this work establishes that targeted MS is a useful approach to support validation of protein expression in studies of the genetics of splicing.
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BibTeX
@misc{lehe2026targeted,
title = {{Targeted proteomics informed by long-read RNA-sequencing reveals protein isoform expression associated with bone mineral density}},
author = {Micah D. Lehe and Vasilii Pavelko and Erin D. Jeffery and Vedavathi Madhu and Larry D. Mesner and Megan D. Schertzer and Abhijit S. Dighe and Charles R. Farber and Gloria M. Sheynkman},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.28.759629},
url = {https://www.biorxiv.org/content/10.64898/2026.10.28.759629v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Targeted proteomics informed by long-read RNA-sequencing reveals protein isoform expression associated with bone mineral density
AU - Micah D. Lehe
AU - Vasilii Pavelko
AU - Erin D. Jeffery
AU - Vedavathi Madhu
AU - Larry D. Mesner
AU - Megan D. Schertzer
AU - Abhijit S. Dighe
AU - Charles R. Farber
AU - Gloria M. Sheynkman
PY - 2026
DA - 2026/10/28
PB - bioRxiv
DO - 10.64898/2026.10.28.759629
UR - https://www.biorxiv.org/content/10.64898/2026.10.28.759629v1
AB - The expanding catalog of sQTLs from projects like GTEx has established that genetic risk for complex traits is frequently mediated through RNA splicing. Yet it is generally unknown whether disease-associated splice events produce stable, distinct protein isoforms and whether they are functionally consequent. Even the deepest shotgun proteomics efforts only capture a minority of annotated splice events; isoform-specific peptides are systematically under-sampled by nature of being lower abundance and intrinsically harder to detect. Here, we implement a genetics-guided targeted mass spectrometry (MS) strategy to detect splice-derived protein isoforms prioritized by sQTL colocalization. Using internal standard parallel reaction monitoring (IS-PRM), we targeted 288 peptides corresponding to 166 putative BMD-associated isoforms in differentiating osteoprogenitor cells and patient-derived primary mesenchymal stem cells (MSCs). Targeted acquisition increased recovery of isoform-informative peptides relative to conventional DDA, rescued splice events that were undetected or sparsely sampled under global acquisition, and confirmed protein-level expression of genetically prioritized isoforms in clinical material. Together, this work establishes that targeted MS is a useful approach to support validation of protein expression in studies of the genetics of splicing.
SN - 2692-8205
ER -