Investigating protein isoforms via proteomics: A feasibility study

Abstract

Alternative splicing (AS) and processing of pre-messenger RNAs explains the discrepancy between the number of genes and proteome complexity in multicellular eukaryotic organisms. However, relatively few alternative protein isoforms have been experimentally identified, particularly at the protein level. In this study, we assess the ability of proteomics to inform on differently spliced protein isoforms in human and four other model eukaryotes. The number of Ensembl-annotated genes for which proteomic data exists that informs on alternative splicing exceeds 33% of the alternately spliced genes in the human and worm genomes. Examining AS in chicken for the first time, we find proteomic support for over 600 genes. However, although peptide identifications support only a small fraction of alternative protein isoforms that are annotated in Ensembl, many more variants are amenable to proteomic identification. There remains a sizeable gap between these existing identifications (10-51% of AS genes) and those that are theoretically feasible (90-99%). We also compare annotations between Swiss-Prot and Ensembl, recommending use of both to maximise coverage of AS. We propose that targeted proteomic experiments using selected reactions and standards are essential to uncover further alternative isoforms and discuss the issues surrounding these strategies.

Discovery and Mass Spectrometric Analysis of Novel Splice-junction Peptides Using RNA-Seq

“…Another important issue in the discovery of alternative splice forms at the protein level is the low number of splicespecific peptides actually identified, an issue that has been revealed by work reported in the literature (17,19,22,24,25,30,31). Part of the reason for the low number of alternative splice variants detected are the technical differences between RNA-Seq and bottom-up proteomics, namely sequence coverage and detection sensitivity.…”

“…Transcripts can be sensitively (Ͻ1 transcript/cell) and completely (100% sequence coverage) characterized, but for proteins, only moderately or highly expressed (Ͼ1 protein molecules/cell) proteins are usually detected and amino acid sequence coverage is typically low (ϳ5-25%). Alternatively spliced proteins are difficult to detect because 1) they have lower cellular abundances than the canonical forms, 2) require at least one splice form-specific peptide for unambiguous detection, likely one spanning a junction or residing in a splice form-specific exon (24), and, 3) the alternative splice variant sequence is sometimes not yet in the database.…”

“…The number of alternative splice forms expected to be detected in a bottom-up proteomics experiment has been estimated using computational approaches (19,22,24). Some authors reported that they identified the expected number of splice-specific peptides while other authors identified far fewer peptides than predicted.…”

“…Initial approaches searched proteomic data against databases containing splice variant sequences and then confirmed the translation of a spliced sequence by detecting a peptide unique to that form (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Other approaches expanded the number of alternatively spliced sequences beyond entries present in databases by constructing exon-exon databases.…”

Proteomic Studies on the Development of the Central Nervous System and Beyond

“…For example, there are currently seventeen differently spliced transcript variants of human brain-derived neurotrophic factor (BDNF) in The National Center for Biotechnology Information (NCBI) RefSeq database (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/ mRNA_Prot/human.rna.fna.gz, May 25, 2010). The proteomic approaches for identifying the splicing variants at the protein level are still under development, although current research has been encouraging, as shown in a recent report by Blakeley [32]. Blakeley et al assessed the ability of proteomics to identify differently spliced protein isoforms in human and four other model eukaryotes and found support for over 600 alternatively spliced (AS) genes in chicken via proteomics.…”