What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

Marcus, Katrin; Lelong, Cécile; Rabilloud, Thierry

doi:10.3390/proteomes8030017

Cited by 49 publications

(31 citation statements)

References 230 publications

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“…We thus used this model to study the changes linked to the adherence status, using a proteomic screen to get a wider appraisal of the phenomena at play. To this purpose, we chose to perform 2D gel-based proteomics, as this proteomic setup is able to detect changes in post-translationally modified protein forms [ 39 ] and thus get closer to the cell physiology [ 40 ]. In the same trend, we chose to validate the proteomic results by functional assays rather than assays based on protein amounts [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

et al. 2021

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Except cells circulating in the bloodstream, most cells in vertebrates are adherent. Studying the repercussions of adherence per se in cell physiology is thus very difficult to carry out, although it plays an important role in cancer biology, e.g. in the metastasis process. In order to study how adherence impacts major cell functions, we used a murine macrophage cell line. Opposite to the monocyte/macrophage system, where adherence is associated with the acquisition of differentiated functions, these cells can be grown in both adherent or suspension conditions without altering their differentiated functions (phagocytosis and inflammation signaling). We used a proteomic approach to cover a large panel of proteins potentially modified by the adherence status. Targeted experiments were carried out to validate the proteomic results, e.g. on metabolic enzymes, mitochondrial and cytoskeletal proteins. The mitochondrial activity was increased in non-adherent cells compared with adherent cells, without differences in glucose consumption. Concerning the cytoskeleton, a rearrangement of the actin organization (filopodia vs sub-cortical network) and of the microtubule network were observed between adherent and non-adherent cells. Taken together, these data show the mechanisms at play for the modification of the cytoskeleton and also modifications of the metabolic activity between adherent and non-adherent cells.

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Section: Discussionmentioning

confidence: 99%

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

et al. 2021

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“…Thus, extensive validation studies would be necessary to rule out false-positive and false-negative results in the published data. Much of this confusion seems likely to arise from a lack of attention to the analysis of proteoforms [ 110 , 111 , 113 , 124 , 134 , 244 , 296 ], which would require a more concerted and detailed effort to identify critical protein species rather than simply cataloging canonical proteins. Certainly, there are data ( Supplementary Table S1 ) consistently identifying at least nine canonical proteins across studies, with the top-down analyses specifically identifying these as particular proteoforms of the generic amino acid sequence (i.e., there is a notable variance between the theoretical and experimentally observed pI and/or MW).…”

Section: Discussionmentioning

confidence: 99%

Proteomics of Multiple Sclerosis: Inherent Issues in Defining the Pathoetiology and Identifying (Early) Biomarkers

Sen

Almuslehi

Shortland

et al. 2021

IJMS

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Multiple Sclerosis (MS) is a demyelinating disease of the human central nervous system having an unconfirmed pathoetiology. Although animal models are used to mimic the pathology and clinical symptoms, no single model successfully replicates the full complexity of MS from its initial clinical identification through disease progression. Most importantly, a lack of preclinical biomarkers is hampering the earliest possible diagnosis and treatment. Notably, the development of rationally targeted therapeutics enabling pre-emptive treatment to halt the disease is also delayed without such biomarkers. Using literature mining and bioinformatic analyses, this review assessed the available proteomic studies of MS patients and animal models to discern (1) whether the models effectively mimic MS; and (2) whether reasonable biomarker candidates have been identified. The implication and necessity of assessing proteoforms and the critical importance of this to identifying rational biomarkers are discussed. Moreover, the challenges of using different proteomic analytical approaches and biological samples are also addressed.

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“…However, it seems to us [69] and to others (e.g., in Zhan et al [70]) that the coupling of high-sensitivity mass spectrometry with 2DGE has a lot to offer in proteomics at the proteoform level. Indeed, high-sensitivity mass spectrometry offers a wealth of peptide information for each 2D spot analyzed.…”

Section: Progress In 2dge Proteomics: Do and Darementioning

confidence: 94%

“…In our opinion, 2DGE proteomics shall be viewed as a mature, efficient, and cost-efficient way to enter the top-down proteomic field, despite the known limitations of 2D gels for the analysis of hydrophobic [67,68], high molecular weight proteins, and proteins with extreme isoelectric points [69]. Although it is quite clear that the resolving power of 2DGE is insufficient compared to the diversity of proteoforms [49,70], 2DGE is an efficient tool for at least a pre-separation of isoforms.…”

Section: Progress In 2dge Proteomics: Do and Darementioning

confidence: 99%

How Do the Different Proteomic Strategies Cope with the Complexity of Biological Regulations in a Multi-Omic World? Critical Appraisal and Suggestions for Improvements

Marcus

Rabilloud

2020

Proteomes

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In this second decade of the 21st century, we are lucky enough to have different types of proteomic analyses at our disposal. Furthermore, other functional omics such as transcriptomics have also undergone major developments, resulting in mature tools. However, choice equals questions, and the major question is how each proteomic strategy is fit for which purpose. The aim of this opinion paper is to reposition the various proteomic strategies in the frame of what is known in terms of biological regulations in order to shed light on the power, limitations, and paths for improvement for the different proteomic setups. This should help biologists to select the best-suited proteomic strategy for their purposes in order not to be driven by raw availability or fashion arguments but rather by the best fitness for purpose. In particular, knowing the limitations of the different proteomic strategies helps in interpreting the results correctly and in devising the validation experiments that should be made downstream of the proteomic analyses.

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What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

Cited by 49 publications

References 230 publications

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

Proteomics of Multiple Sclerosis: Inherent Issues in Defining the Pathoetiology and Identifying (Early) Biomarkers

How Do the Different Proteomic Strategies Cope with the Complexity of Biological Regulations in a Multi-Omic World? Critical Appraisal and Suggestions for Improvements

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