Identification and Quantification of Proteoforms by Mass Spectrometry

Abstract

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.

Proteomics and Metabolomics in Kidney Disease, including Insights into Etiology, Treatment, and Prevention

“…The NIDDK Kidney Precision Medicine Project (www.kpmp.org) is anticipated to provide much-needed human kidney tissues for biomarker research, and this program and more mechanistic investigation in model systems will be critical to determine both why select proteins or metabolites are altered in kidney disease and whether they play a causal role in disease pathogenesis. Recent advances in identifying post-translational modifications of proteins will enhance our understanding of the proteome in kidney disease (49). In parallel, the integration of genomics with proteomics and metabolomics may permit Mendelian randomization-based approaches to test whether select markers lie in causal pathways (50).…”

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

“…The quantitative analysis of intact proteins (a so‐called top‐down approach, which bypasses enzymatic digestion and introduces intact proteins directly into the mass spectrometer), is becoming more feasible every year and has experienced tremendous growth over the last several years [3,8–11]. A major obstacle in top‐down proteomics is sample complexity, as multiple proteoforms exist for each protein.…”

FLASHDeconv: Ultrafast, high-quality feature deconvolution for top-down proteomics

“…Top-down (TD) proteomics has gained a lot of momentum for in-depth protein characterization and protein species analytics [1][2][3][4][5][6][7] . In contrast to bottom-up (BU) proteomics, where proteins are enzymatically digested and actually peptides are analyzed, the TD approach allows for the analysis of intact proteoforms (protein species arising from the same gene product via splice variants, genomic variation, post-translational modifications, degradation, etc.)…”

“…In contrast to bottom-up (BU) proteomics, where proteins are enzymatically digested and actually peptides are analyzed, the TD approach allows for the analysis of intact proteoforms (protein species arising from the same gene product via splice variants, genomic variation, post-translational modifications, degradation, etc.) 6,8 . Due to the high mass of the analytes, however, in mass spectrometry (MS)-based TD approach (TD-MS), a single protein species can result in multiple MS features with different charge states and isotopic envelopes, making the signal structure highly complex and, in turn, the accurate determination of proteoform masses challenging 9 .…”

“…Current MS instrumentation and experimental protocols enable more complex analyses using TD proteomics, but in turn yield equally complex and large data sets 1,2,6,7,10,12 . Hence, both quality and runtime of deconvolution algorithms have become an issue.…”