scholarly journals Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

2012 ◽  
Vol 2012 ◽  
pp. 1-17 ◽  
Author(s):  
Cosette Abdallah ◽  
Eliane Dumas-Gaudot ◽  
Jenny Renaut ◽  
Kjell Sergeant
Keyword(s):  
Quantitative Proteomics ◽  
Protein Extraction ◽  
State Of The Art ◽  
Rapid Expansion ◽  
Protein Abundance ◽  
Label Free ◽  
Genome Sequences ◽  
Two Dimensional Gel Electrophoresis ◽  
Green Tissue ◽  
Peptide Fractionation

Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.

scholarly journals Proteomic characterization of pilot scale hot-water extracts from the industrial carrageenan red seaweed Eucheuma denticulatum

2020 ◽  
Author(s):  
Simon Gregersen ◽  
Margarita Pertseva ◽  
Paolo Marcatili ◽  
Susan Løvstad Holdt ◽  
Charlotte Jacobsen ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
De Novo ◽  
Protein Extraction ◽  
Transcriptome Assembly ◽  
Protein Composition ◽  
Pilot Scale ◽  
Extracellular Proteins ◽  
Hot Water ◽  
Label Free

AbstractSeaweeds have a long history as a resource for polysaccharides/hydrocolloids extraction for use in the food industry due to their functionality as stabilizing agents. In addition to the carbohydrate content, seaweeds also contains a significant amount of protein, which may find application in food and feed. Here, we present a novel combination of transcriptomics, proteomics, and bioinformatics to determine the protein composition in two pilot-scale extracts from Eucheuma denticilatum (Spinosum) obtained via hot-water extraction. The extracts were characterized by qualitative and quantitative proteomics using LC-MS/MS and a de-novo transcriptome assembly for construction of a novel proteome. Using label-free, relative quantification, we were able to identify the most abundant proteins in the extracts and determined that the majority of quantified protein in the extracts (>75%) is constituted by merely three previously uncharacterized proteins. Putative subcellular localization for the quantified proteins was determined by bioinformatic prediction, and by correlating with the expected copy number from the transcriptome analysis, we determined that the extracts were highly enriched in extracellular proteins. This implies that the method predominantly extracts extracellular proteins, and thus appear ineffective for cellular disruption and subsequent release of intracellular proteins. Ultimately, this study highlight the power of quantitative proteomics as a novel tool for characterization of alternative protein sources intended for use in foods. Additionally, the study showcases the potential of proteomics for evaluation of protein extraction methods and as powerful tool in the development of an efficient extraction process.

scholarly journals Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3591
Author(s):  
Pratiti Bhadra ◽  
Stefan Schorr ◽  
Monika Lerner ◽  
Duy Nguyen ◽  
Johanna Dudek ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Secretory Pathway ◽  
Human Cells ◽  
Ribosome Profiling ◽  
Protein Abundance ◽  
Label Free ◽  
Differential Protein ◽  
Cellular Assays ◽  
Pros And Cons ◽  
Er Membrane

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

Development of Algorithms for Mass Spectrometry-based Label-free Quantitative Proteomics*

2011 ◽  
Vol 38 (6) ◽  
pp. 506-518 ◽  
Author(s):  
Wei ZHANG ◽  
Ji-Yang ZHANG ◽  
Hui LIU ◽  
Han-Chang SUN ◽  
Chang-Ming XU ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Label Free

scholarly journals Temporal Quantitative Proteomics Analysis of Neuroblastoma Cells Treated with Bovine Milk-Derived Extracellular Vesicles Highlights the Anti-Proliferative Properties of Milk-Derived Extracellular Vesicles

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 750
Author(s):  
Pamali Fonseka ◽  
Taeyoung Kang ◽  
Sing Chee ◽  
Sai V. Chitti ◽  
Rahul Sanwlani ◽  
...  
Keyword(s):  
High Risk ◽  
Extracellular Vesicles ◽  
Quantitative Proteomics ◽  
Cell Metabolism ◽  
Bovine Milk ◽  
Neuroblastoma Cells ◽  
Treatment Strategies ◽  
Treatment Resistance ◽  
Label Free ◽  
The Impact

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

scholarly journals Label-free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5

PROTEOMICS ◽  
2011 ◽  
Vol 11 (8) ◽  
pp. 1508-1516 ◽  
Author(s):  
Dana V. Skarra ◽  
Marilyn Goudreault ◽  
Hyungwon Choi ◽  
Michael Mullin ◽  
Alexey I. Nesvizhskii ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Quantitative Proteomics ◽  
Label Free ◽  
Protein Phosphatase 5 ◽  
Interactome Mapping
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