Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2

In eukaryotes, alternative pre-mRNA
splicing allows a single gene
to encode different protein isoforms that function in many biological
processes, and they are used as biomarkers or therapeutic targets
for diseases. Although protein isoforms in the human genome are well
annotated, we speculate that some low-abundance protein isoforms may
still be under-annotated because most genes have a primary coding
product and alternative protein isoforms tend to be under-expressed.
A peptide coencoded by a novel exon and an annotated exon separated
by an intron is known as a novel junction peptide. In the absence
of known transcripts and homologous proteins, traditional whole-genome
six-frame translation-based proteogenomics cannot identify novel junction
peptides, and it cannot capture novel alternative splice sites. In
this article, we first propose a strategy and tool for identifying
novel junction peptides, called CJunction, which we then integrate
into a proteogenomics process specifically designed for novel protein
isoform discovery and apply to the analysis of a deep-coverage HeLa
mass spectrometry data set with identifier PXD004452 in ProteomeXchange.
We succeeded in identifying and validating three novel protein isoforms
of two functionally important genes, NHSL1 (causative gene of Nance-Horan
syndrome) and EEF1B2 (translation elongation factor), which validate
our hypothesis. These novel protein isoforms have significant sequence
differences from the annotated gene-coding products introduced by
the novel N-terminal, suggesting that they may play importantly different
functions.