Fabrication of a monolithic, macroporous diallyl maleate-based material and its application for fast separation of intact proteins from human plasma with reversed-phase chromatography

Lan, Dandan; Bai, Ligai; Liu, Haiyan; Guo, Hongwei; Yan, Hongyuan

doi:10.1016/j.chroma.2019.01.026

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…As for bottom-up proteomics, RP UPLC remains the method of choice for online separation because of its compatibility with (n)ESI. There have been substantive improvements in the separation of proteins for TDP by RP chromatography over recent years due to the development of protein-specific UPLC (and HPLC) media, with monolithic columns functionalized with short (C 4 –C 8 ) alkyl chains becoming popular. − As important as RP chromatography has and will continue to be for TDP, separation based on hydrophobicity inherently limits the ability to separate large proteoforms that may differ by small modifications, e.g., oxidation, acetylation, phosphorylation. In this regard, (online) capillary electrophoresis (CE) has shown some promise, proving to be an orthogonal separation strategy to RP chromatography in its ability to separate analytes for identification .…”

Section: Proteoform Separationthe Issue Of Chimeric Spectramentioning

confidence: 99%

Top-Down Proteomics and the Challenges of True Proteoform Characterization

Po,

Eyers

2023

J. Proteome Res.

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Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires that both the base protein sequence be defined and any mass shifts identified, ideally localizing their positions within the protein sequence. Being able to fully elucidate proteoform profiles lends insight into characterizing proteoform-unique roles, and is a crucial aspect of defining protein structure−function relationships and the specific roles of different (combinations of) protein modifications. However, defining and pinpointing protein posttranslational modifications (PTMs) on intact proteins remains a challenge. Characterization of (heavily) modified proteins (>∼30 kDa) remains problematic, especially when they exist in a population of similarly modified, or kindred, proteoforms. This issue is compounded as the number of modifications increases, and thus the number of theoretical combinations. Here, we present our perspective on the challenges of analyzing kindred proteoform populations, focusing on annotation of protein modifications on an "average" protein. Furthermore, we discuss the technical requirements to obtain high quality fragmentation spectral data to robustly define site-specific PTMs, and the fact that this is tempered by the time requirements necessary to separate proteoforms in advance of mass spectrometry analysis.

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Section: Proteoform Separationthe Issue Of Chimeric Spectramentioning

confidence: 99%

Top-Down Proteomics and the Challenges of True Proteoform Characterization

Po,

Eyers

2023

J. Proteome Res.

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“…Mann et al (2017) evidenced the effects of high‐pressure monolithic separations on the retention properties of intact proteins, suggesting that reproducibility is optimized by operating <200 bar. Lan et al (2019) reported a microporous diallyl maleate‐based monolithic packing material, which they employed to separate plasma proteins in 15 min. Liang et al (2019) used one step sol‐gel condensation with C8 functionalization to construct a bridged hybrid monolithic column.…”

Section: Fractionation Approaches For Top‐down Proteomicsmentioning

confidence: 99%

Recent advances in top‐down proteome sample processing ahead of MS analysis

Nickerson

Baghalabadi

Rajendran

et al. 2021

Mass Spectrometry Reviews

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Top‐down proteomics is emerging as a preferred approach to investigate biological systems, with objectives ranging from the detailed assessment of a single protein therapeutic, to the complete characterization of every possible protein including their modifications, which define the human proteoform. Given the controlling influence of protein modifications on their biological function, understanding how gene products manifest or respond to disease is most precisely achieved by characterization at the intact protein level. Top‐down mass spectrometry (MS) analysis of proteins entails unique challenges associated with processing whole proteins while maintaining their integrity throughout the processes of extraction, enrichment, purification, and fractionation. Recent advances in each of these critical front‐end preparation processes, including minimalistic workflows, have greatly expanded the capacity of MS for top‐down proteome analysis. Acknowledging the many contributions in MS technology and sample processing, the present review aims to highlight the diverse strategies that have forged a pathway for top‐down proteomics. We comprehensively discuss the evolution of front‐end workflows that today facilitate optimal characterization of proteoform‐driven biology, including a brief description of the clinical applications that have motivated these impactful contributions.

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