Recent Advances in Multidimensional Separation for Proteome Analysis

Yuan, Huan; Jiang, Bo; Zhao, Baofeng; Zhang, Lihua; Zhang, Yukui

doi:10.1021/acs.analchem.8b04894

Cited by 39 publications

(44 citation statements)

References 144 publications

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“…Thus, there is a great need to develop high-resolution separation systems to improve proteome coverage and to enable the detection of low-abundance proteins. 12…”

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confidence: 99%

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

et al. 2019

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Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-μm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 μL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl β-D-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ~49,000–94,000 unique peptides, corresponding to ~6,700–8,300 protein groups using only 100–1000 ng of HeLa tryptic digest (equivalent to ~500–5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ~20,000 phosphopeptides from 100 μg of MCF-7 cell lysate.

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“…Thus, there is a great need to develop high-resolution separation systems to improve proteome coverage and to enable the detection of low-abundance proteins. 12…”

mentioning

confidence: 99%

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

et al. 2019

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“…2D Separations: Achieving deep proteome coverage of >5000 protein groups generally requires a multidimensional separation that provides greater peak capacity than that of a one dimensional separation. However, fractionation schemes typically lose significant amounts of sample to the collection vessels between the two separation dimensions, thus posing a significant challenge for lowinput proteomics (61). To address this, we recently developed nanowell-mediated 2D LC in which the same glass chips used for nanoPOTS sample preparation served as fraction collectors between two dimensions of nanoLC (17) ( Figure 2D).…”

Section: Progressmentioning

confidence: 99%

Single-cell Proteomics: Progress and Prospects

Kelly

2020

Molecular & Cellular Proteomics

261

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MS-based proteome profiling has become increasingly comprehensive and quantitative, yet a persistent shortcoming has been the relatively large samples required to achieve an in-depth measurement. Such bulk samples, typically comprising thousands of cells or more, provide a population average and obscure important cellular heterogeneity. Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells, a level of coverage that required an input of thousands of cells just a few years ago. This review discusses important factors and parameters that should be optimized across the workflow for single-cell and other low-input measurements. It also highlights recent developments that have advanced the field and opportunities for further development.

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“…The sample loss is thereby considerably low. Online strategies sometimes decrease the overall time from sample preparation to analysis [19,42]. Thus, each strategy has some advantages and drawbacks.…”

Section: Comparison Of Offline and Online Setupsmentioning

confidence: 99%

“…Some studies have performed quantitative proteomics with MD-LC strategies to achieve a deep coverage proteome quantification [6,42,44]. It is of great benefit for clinical applications, particularly for the discovery of biomarkers [91].…”

Section: Author's Outlook and Concluding Remarksmentioning

confidence: 99%

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Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics

Duong

Park

Lee

2020

IJMS

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Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

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Recent Advances in Multidimensional Separation for Proteome Analysis

Cited by 39 publications

References 144 publications

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

Single-cell Proteomics: Progress and Prospects

Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics

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