Enhanced Discovery of Alternative Proteins (AltProts) in Mouse Cardiac Development Using Data-Independent Acquisition (DIA) Proteomics

Alternative
proteins (AltProts) are a class of proteins encoded
by DNA sequences previously classified as noncoding. Despite their
historically being overlooked, recent studies have highlighted their
widespread presence and distinctive biological roles. So far, direct
detection of AltProt has been relying on data-dependent acquisition
(DDA) mass spectrometry (MS). However, data-independent acquisition
(DIA) MS, a method that is rapidly gaining popularity for the analysis
of canonical proteins, has seen limited application in AltProt research,
largely due to the complexities involved in constructing DIA libraries.
In this study, we present a novel DIA workflow that leverages a fragmentation
spectra predictor for the efficient construction of DIA libraries,
significantly enhancing the detection of AltProts. Our method achieved
a 2-fold increase in the identification of AltProts and a 50% reduction
in missing values compared to DDA. We conducted a comprehensive comparison
of four AltProt databases, four DIA-library construction strategies,
and three analytical software tools to establish an optimal workflow
for AltProt analysis. Utilizing this workflow, we investigated the
mouse heart development process and identified over 50 AltProts with
differential expression between embryonic and adult heart tissues.
Over 30 unannotated mouse AltProts were validated, including ASDURF,
which played a crucial role in cardiac development. Our findings not
only provide a practical workflow for MS-based AltProt analysis but
also reveal novel AltProts with potential significance in biological
functions.