Lights and shadows of proteomic technologies for the study of protein species including isoforms, splicing variants and protein post‐translational modifications

Abstract

Recent reviews pinpointed the enormous diversity of proteins found in living organisms, especially in higher eukaryotes. Protein diversity is driven through three main processes: first, at deoxyribonucleic acid (DNA) level (i.e. gene polymorphisms), second, at precursor messenger ribonucleic acid (pre‐mRNA) or messenger ribonucleic acid (mRNA) level (i.e. alternative splicing, also termed as differential splicing) and, finally, at the protein level (i.e. PTM). Current proteomic technologies allow the identification, characterization and quantitation of up to several thousands of proteins in a single experiment. Nevertheless, the identification and characterization of protein species using these technologies are still hampered. Here, we review the use of the terms “protein species” and “protein isoform.” We evidence that the appropriate selection of the database used for searches can impede or facilitate the identification of protein species. We also describe examples where protein identification search engines systematically fail in the attribution of protein species. We briefly review the characterization of protein species using proteomic technologies including gel‐based, gel‐free, bottom‐up and top‐down analysis and discuss their limitations. As an example, we discuss the theoretical characterization of the two human choline kinase species, α‐1 and α‐2, sharing the same catalytic activity but generated by alternative splicing on CHKA gene.

New alternatively spliced variant of prostate-specific membrane antigen PSM-E suppresses the proliferation, migration and invasiveness of prostate cancer cells

“…Recent genome wide analysis of alternative splicing indicates that as much as 70% of human genes may have alternative spliced forms (17,20,21). Changes in splice site selection and the production of splice variants have been observed in various types of cancer and may affect tumor progression and susceptibility to cancer.…”

Approaches for the study of cancer: towards the integration of genomics, proteomics and metabolomics

“…On top of this, other sources of complexity include mechanisms leading to the generation of molecular diversity and functional regulation. As an example, the generation of protein species [1] is driven by three main processes: fi rst, at deoxyribonucleic acid (DNA) level (i.e., gene polymorphisms), second, at precursor messenger ribonucleic acid (pre-mRNA) or messenger ribonucleic acid (mRNA) level (i.e., alternative splicing, also termed as differential splicing) and, fi nally, at the protein level (i.e., posttranslational modifi cations) (e.g., [1] for review).…”

“…Whilst CHKA is exclusively attributed to choline kinase genes from different organisms (including humans), CHK and CKI can be used to denote checkpoint kinases or casein kinase I, respectively. Regarding protein annotation, one protein may correspond to more than one accession number both in public databases and in the literature [1,2]. Moreover, protein accession numbers and descriptions may vary with time as long as databases are annotated and released.…”

Automated Mass Spectrometry–Based Functional Assay for the Routine Analysis of the Secretome

“…[34][35][36] These two different approaches are conventionally or historically performed on different instruments (i.e., mass spectrometers that have different ionization sources). In top-down secretomics, the preferred mass spectrometer has a matrix-assisted laser desorption ionization source (MALDI source; the mass spectrometer is MALDI-MS), whereas in bottom-up secretomics, the preferred mass spectrometer has an electrospray ionization (ESI) source and the mass spectrometer is named ESI-MS. [37][38][39] If the MS is coupled with high-performance liquid chromatography (HPLC), then the MS system is HPLC-ESI-MS or LC-MS. If the MS is a tandem MS, then the LC-MS is an LC-MS/MS.…”