scholarly journals Complexome Profiling: Assembly and Remodeling of Protein Complexes

2021 ◽  
Vol 22 (15) ◽  
pp. 7809
Author(s):  
Ilka Wittig ◽  
Pedro Felipe Malacarne
Keyword(s):  
Mass Spectrometry ◽  
Density Gradient Centrifugation ◽  
Protein Complexes ◽  
Gradient Centrifugation ◽  
Protein Assemblies ◽  
Quantitative Mass Spectrometry ◽  
Macromolecular Complexes ◽  
A Cell ◽  
Health And Disease ◽  
Native Gel

Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.

scholarly journals Emerging mass spectrometry-based proteomics methodologies for novel biomedical applications

2020 ◽  
Vol 48 (5) ◽  
pp. 1953-1966
Author(s):  
Lindsay K. Pino ◽  
Jacob Rose ◽  
Amy O'Broin ◽  
Samah Shah ◽  
Birgit Schilling
Keyword(s):  
Mass Spectrometry ◽  
Human Health ◽  
Protein Complexes ◽  
Protein Quantification ◽  
Intact Protein ◽  
Protein Signaling ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Health And Disease ◽  
Proteomics Research

Research into the basic biology of human health and disease, as well as translational human research and clinical applications, all benefit from the growing accessibility and versatility of mass spectrometry (MS)-based proteomics. Although once limited in throughput and sensitivity, proteomic studies have quickly grown in scope and scale over the last decade due to significant advances in instrumentation, computational approaches, and bio-sample preparation. Here, we review these latest developments in MS and highlight how these techniques are used to study the mechanisms, diagnosis, and treatment of human diseases. We first describe recent groundbreaking technological advancements for MS-based proteomics, including novel data acquisition techniques and protein quantification approaches. Next, we describe innovations that enable the unprecedented depth of coverage in protein signaling and spatiotemporal protein distributions, including studies of post-translational modifications, protein turnover, and single-cell proteomics. Finally, we explore new workflows to investigate protein complexes and structures, and we present new approaches for protein–protein interaction studies and intact protein or top-down MS. While these approaches are only recently incipient, we anticipate that their use in biomedical MS proteomics research will offer actionable discoveries for the improvement of human health.

scholarly journals Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1289
Author(s):  
Fabian Henneberg ◽  
Ashwin Chari
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Macromolecular Complex ◽  
Biological Macromolecules ◽  
Chromatographic Purification ◽  
Complex Interplay ◽  
Macromolecular Complexes ◽  
Functional Interactions ◽  
Biotechnological Processes ◽  
Essential Prerequisite

A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic interrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes.

scholarly journals Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

2008 ◽  
Vol 183 (2) ◽  
pp. 223-239 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Séverine Boulon ◽  
Yun Wah Lam ◽  
Roby Urcia ◽  
François-Michel Boisvert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Cell Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Specific Protein ◽  
Protein Interaction Data ◽  
Quantitative Mass Spectrometry ◽  
Cell Extracts ◽  
Interaction Partners

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

scholarly journals Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

2021 ◽  
Author(s):  
Christina Wichmann ◽  
Petra Rösch ◽  
Jürgen Popp
Keyword(s):  
Raman Spectroscopy ◽  
Bronchoalveolar Lavage ◽  
Density Gradient ◽  
Bronchoalveolar Lavage Fluid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Lavage Fluid ◽  
Biochemical Properties ◽  
Low Concentrations ◽  
A Cell

AbstractRaman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix.

A dolichol acyltransferase present in rat and human postheparin plasma

1992 ◽  
Vol 70 (6) ◽  
pp. 470-474 ◽  
Author(s):  
P. Sindelar ◽  
C. Valtersson
Keyword(s):  
Cell Surface ◽  
Molecular Mass ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
High Molecular Mass ◽  
Gel Chromatography ◽  
Heat Treated ◽  
A Cell ◽  
Small Unilamellar Vesicles ◽  
Plasma Heparin

Incubation of small unilamellar vesicles consisting of dioleoyl phosphatidylcholine – dioleoyl phosphatidylethanolamine (3:1) and 2 mol% [3H]dolichol-19 with postheparin plasma from rat resulted in the formation of dolichyl oleate. Normal plasma or heat-treated postheparin plasma contained no activity and, hence, the results indicate the presence of a cell surface associated dolichol acyltransferase that can be released into the blood by heparin. The reaction is strongly stimulated by phosphatidylethanolamine and Ca2+, whereas no stimulation with triglycerides or acyl-CoA was observed. Together with the fact that the only product formed was dolichyl oleate, these results strongly suggest that a transacylation mechanism from the phospholipids to dolichol is operative in the liposomes. Gel chromatography of postheparin plasma yielded a molecular mass of about 350 kilodaltons for the active enzyme and density gradient centrifugation indicated that this high molecular mass complex consists mainly of proteins. Finally, we conclude that this enzyme is not unique to the rat, but is also present in human postheparin plasma.Key words: phospholipids, dolichol, plasma, heparin, acyltransferase(s).

scholarly journals Identification of Cells in Primate Bone Marrow Resembling the Hemopoietic Stem Cell in the Mouse

Blood ◽  
1973 ◽  
Vol 42 (2) ◽  
pp. 195-208 ◽  
Author(s):  
K. A. Dicke ◽  
M. J. van Noord ◽  
B. Maat ◽  
U. W. Schaefer ◽  
D. W. van Bekkum
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Staining Method ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Hemopoietic Stem Cell ◽  
Vitro Assay ◽  
A Cell ◽  
Unit Culture

Abstract The colony-forming unit culture (CFU-C) in the thin-layer agar colony technique is considered to be representative for hemopoietic stem cells (HSC), according to our studies in mouse and monkey bone marrow. Using this in vitro assay as a guide, stem cell concentrates were prepared from monkey and human bone marrow by repeated density gradient centrifugation. The number of CFU-C could be enriched up to 70-100-fold. In such concentrated CFU-C suspensions, a cell, morphologically identical with the hemopoietic stem cell in the mouse (MSCLC, mouse stem cell-like cell) was frequently observed, using a May-Grünwald-Giemsa (MGG) staining method and electron microscope techniques. In MGG-stained preparations, the MSCLC superficially resembles the small lymphocyte; therefore, a staining method has been described, the polychrome procedure, by which both cell populations could be clearly distinguished. Since a fair correlation exists between the number of MSCLC and the number of CFU-C in a variety of primate hemopoietic suspensions, we concluded that the MSCLC might be a good candidate for being the HSC in monkeys and man.

scholarly journals Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics

2012 ◽  
Vol 41 (1) ◽  
pp. e28-e28 ◽  
Author(s):  
Arne H. Smits ◽  
Pascal W. T. C. Jansen ◽  
Ina Poser ◽  
Anthony A. Hyman ◽  
Michiel Vermeulen
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Label Free ◽  
Quantitative Mass Spectrometry

scholarly journals Dissection of affinity captured LINE-1 macromolecular complexes

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Martin S Taylor ◽  
Ilya Altukhov ◽  
Kelly R Molloy ◽  
Paolo Mita ◽  
Hua Jiang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mobile Genetic Element ◽  
Host Factors ◽  
Genetic Element ◽  
Quantitative Mass Spectrometry ◽  
Host Proteins ◽  
Macromolecular Complexes ◽  
Human Genomes ◽  
Long Interspersed Nuclear Element ◽  
Differential Affinity

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.

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