Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma

Li, Na; Zhou, Yue; Wang, Jifeng; Niu, Lili; Zhang, Qing; Sun, Lang; Ding, Xiang; Guo, Xiaojing; Xie, Zenghong; Zhu, Nali; Zhang, Mengmeng; Chen, Xiu-Lan; Cai, Tao; Yang, Fuquan

doi:10.1021/acs.jproteome.0c00232

Cited by 19 publications

(26 citation statements)

References 52 publications

(71 reference statements)

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“…This method is fast, easy to perform and can identify many unique peptides in serum and plasma. , However, peptides with molecular weights close to the cutoff point may be lost, such as apolipoproteins, and some “sticky” lipophilic peptides might be lost by binding to the membranes. While there is also a possibility of contamination from higher molecular weight fractions, Li showed that most of the peptide content was lost using a centrifugal MWCO device and that a simple precipitation approach was superior in its recovery of low molecular weight components of plasma.…”

Section: Methodologies For Peptidomics Analysismentioning

confidence: 99%

“…These have been used to efficiently recover peptides from biological samples . A recently published method used sequential plasma precipitation and delipidation to enrich low molecular weight plasma proteins for measurement . Using this combination of extraction methods and methyl tert -butyl ether, smaller proteins and peptides were extracted from human plasma samples.…”

Section: Methodologies For Peptidomics Analysismentioning

confidence: 99%

“…67 A recently published method used sequential plasma precipitation and delipidation to enrich low molecular weight plasma proteins for measurement. 61 Using this combination of extraction methods and methyl tert-butyl ether, smaller proteins and peptides were extracted from human plasma samples. Visualized by SDS-PAGE, this approach was found to have a similar recovery of peptides when compared to other combined plasma precipitation and SPE methods.…”

Section: ■ Methodologies For Peptidomics Analysis Extraction Approachesmentioning

confidence: 99%

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Peptidomics: A Review of Clinical Applications and Methodologies

et al. 2021

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Improvements in both liquid chromatography (LC) and mass spectrometry (MS) instrumentation have greatly enhanced proteomic and small molecule metabolomic analysis in recent years. Less focus has been on the improved capability to detect and quantify small bioactive peptides, even though the exact sequences of the peptide species produced can have important biological consequences. Endogenous bioactive peptide hormones, for example, are generated by the targeted and regulated cleavage of peptides from their prohormone sequence. This process may include organ specific variants, as proglucagon is converted to glucagon in the pancreas but glucagon-like peptide-1 (GLP-1) in the small intestine, with glucagon raising, whereas GLP-1, as an incretin, lowering blood glucose. Therefore, peptidomics workflows must preserve the structure of the processed peptide products to prevent the misidentification of ambiguous peptide species. The poor in vivo and in vitro stability of peptides in biological matrices is a major factor that needs to be considered when developing methods to study them. The bioinformatic analysis of peptidomics data sets requires the inclusion of specific posttranslational modifications, which are critical for the function of many bioactive peptides. This review aims to discuss and contrast the various extraction, analytical, and bioinformatics approaches used for human peptidomics studies in a multitude of matrices.

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Section: Methodologies For Peptidomics Analysismentioning

confidence: 99%

Section: Methodologies For Peptidomics Analysismentioning

confidence: 99%

Section: ■ Methodologies For Peptidomics Analysis Extraction Approachesmentioning

confidence: 99%

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Peptidomics: A Review of Clinical Applications and Methodologies

et al. 2021

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“…For isolation of microproteins from whole plasma, the low molecular weight fraction of plasma was collected using a sequential precipitation and de‐lipidation (1) method developed for the efficient enrichment of the low molecular weight proteins/peptides from plasma based on methyl‐tert‐butyl ether/methanol/water systems (Li et al., 2020). Briefly, a single‐phase solvent system MTBE/methanol/water (5:3:1, v/v) and a two‐phase solvent system MTBE/methanol/water (5:1:1, v/v) were applied for sequential precipitation and de‐lipidation of plasma.…”

Section: Methodsmentioning

confidence: 99%

“…After centrifugation at 20,000 g for 20 min at 4°C in a bench-top centrifuge, the supernatant was filtered through a 30 kD MWCO filter (Millipore) and the flow through was collected and evaporated until dry by vacuum centrifugation at 4°C. The pellet was then dissolved in 8 M urea/ 100 mM NH 4 HCO 3 and stored at -80 • C. For isolation of microproteins from whole plasma, the low molecular weight fraction of plasma was collected using a sequential precipitation and de-lipidation (1) method developed for the efficient enrichment of the low molecular weight proteins/peptides from plasma based on methyl-tert-butyl ether/methanol/water systems (Li et al, 2020). Briefly, a single-phase solvent system MTBE/methanol/water (5:3:1, v/v) and a two-phase solvent system MTBE/methanol/water (5:1:1, v/v) were applied for sequential precipitation and de-lipidation of plasma.…”

Section:  Isolation Of Microproteins From Ev-free Plasma and Whole Plasmamentioning

confidence: 99%

LncRNA‐encoded microproteins: A new form of cargo in cell culture‐derived and circulating extracellular vesicles

Cai

Zhang

et al. 2021

J of Extracellular Vesicle

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Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EVmediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma. K E Y WO R D S cancer, extracellular vesicles (EVs), lncRNA-encoded microproteins, long noncoding RNA (lncRNA), small open reading frames (smORFs)This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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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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Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma

Cited by 19 publications

References 52 publications

Peptidomics: A Review of Clinical Applications and Methodologies

Peptidomics: A Review of Clinical Applications and Methodologies

LncRNA‐encoded microproteins: A new form of cargo in cell culture‐derived and circulating extracellular vesicles

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

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