Proteins and Proteoforms: New Separation Challenges

Regnier, Fred E.; Kim, Jinhee

doi:10.1021/acs.analchem.7b05007

Cited by 34 publications

(31 citation statements)

References 156 publications

(275 reference statements)

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“…mAbs selected against phosphoserine/threonine containing proteins (which represent > 99% of all phosphoproteins) [66] suffer from high costs, low specificity, and batch-to-batch variability [62]. Importantly, because affinity ligands are generally designed to select for broader protein families (proteoforms) and are not necessarily limited to single analytes or epitopes such as mAbs, the use of a high quality phospho-specific affinity ligand allows for a global and deep selection of phosphoproteins without bias [30,31,62,67], as demonstrated above in the enrichment of multiple phosphoproteins. Furthermore, due to the easily modifiable amine-coated surface, this nanoproteomics platform can be easily furnished with other affinity ligands, thereby enabling a broad scope of enrichment strategies for other low abundance proteins in general and other biological applications.…”

Section: Resultsmentioning

confidence: 99%

Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: Enrichment of the human heart phosphoproteome

et al. 2019

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A reproducible synthetic strategy was developed for facile large-scale (200 mg) synthesis of surface silanized magnetite (Fe3O4) nanoparticles (NPs) for biological applications. After further coupling a phosphate-specific affinity ligand, these functionalized magnetic NPs were used for the highly specific enrichment of phosphoproteins from a complex biological mixture. Moreover, correlating the surface silane density of the silanized magnetite NPs to their resultant enrichment performance established a simple and reliable quality assurance control to ensure reproducible synthesis of these NPs routinely in large scale and optimal phosphoprotein enrichment performance from batch-to-batch. Furthermore, by successful exploitation of a top-down phosphoproteomics strategy that integrates this high throughput nanoproteomics platform with online liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we were able to specifically enrich, identify, and characterize endogenous phosphoproteins from highly complex human cardiac tissue homogenate. This nanoproteomics platform possesses a unique combination of scalability, specificity, reproducibility, and efficiency for the capture and enrichment of low abundance proteins in general, thereby enabling downstream proteomics applications.

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Section: Resultsmentioning

confidence: 99%

Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: Enrichment of the human heart phosphoproteome

et al. 2019

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“…An organic solvent is used in loading HILIC columns to drive hydrophilic portions of proteins to interact with a hydrophilic stationary phase. Elution using a gradient from an organic solvent to an aqueous buffer allows desorption and elution of proteins from the column [66][67][68]. HILIC is MS-compatible LC technique for protein analysis.…”

Section: Hydrophilic Interaction Chromatography (Hilic)mentioning

confidence: 99%

Proteoforms: General Concepts and Methodological Process for Identification

Araújo¹,

Machado²

2020

Proteoforms - Concept and Applications in Medical Sciences

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The term proteoform is used to denote all the molecular forms in which the protein product of a single gene can be found. The most frequent processes that lead to transcript modification and the biological implications of these changes observed in the final protein product will be discussed. Proteoforms arising from genetic variations, alternatively spliced RNA transcripts and post-translational modifications will be commented. This chapter will present an evolution of the techniques used to identify the proteoforms and the importance of this identification for understanding of biological processes. This chapter highlights the fundamental concepts in the field of top-down mass spectrometry (TDMS), and provides numerous examples for the use of knowledge obtained from the identification of proteoforms. The identification of mutant proteins is one of the emerging areas of proteogenomics and has the potential to recognize novel disease biomarkers and may point to useful targets for identification of therapeutic approaches.

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“…Despite the lower number of components of the protein samples, when the proteins are not proteolyzed, a separation and solubilization step is essential [17]. The solubilization of proteins often requires the addition of surfactants that can interact with stationary chromatography phases and limit the protein's ionization efficiency in MS. To extract and solubilize proteins, the most common surfactant is sodium dodecyl sulfate (SDS), although it is poorly tolerated in MS and LC [165] because it could not be effectively replaced.…”

Section: Purification and Separation Of Proteinsmentioning

confidence: 99%

“…Two decades later, proteomics analysis reached a stage where the quantitative analysis of functional proteoforms [16], as well as the identification of each modification site, is a major objective [17,18]. Each variant of a given primary sequence is the result of many correlated phenomena.…”

mentioning

confidence: 99%

“…The idea is not new [37], but the implementation of a combination of bottom-up, top-down, and middle-down (using limited proteolysis) strategies always remains an analytical challenge [38]. Despite this, the LC-MS analysis of the intact protein is currently considered as a complementary and essential analytical approach [17,39]. Indeed, our goal is the characterization of functional proteoforms.…”

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confidence: 99%

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