scholarly journals Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components

Proteomes ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 18 ◽  
Author(s):  
Mateusz Smolarz ◽  
Monika Pietrowska ◽  
Natalia Matysiak ◽  
Łukasz Mielańczyk ◽  
Piotr Widłak
Keyword(s):  
Human Serum ◽  
Serum Proteins ◽  
Size Exclusion ◽  
Simple Method ◽  
Exclusion Chromatography ◽  
Specific Proteins ◽  
Bona Fide ◽  
Shotgun Approach ◽  
Related Proteins ◽  
Serum Components

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

scholarly journals Characterization of DNA nanostructure stability by size exclusion chromatography

2021 ◽  
Author(s):  
Nicole Langlois ◽  
Heather Clark
Keyword(s):  
Human Serum ◽  
Size Exclusion Chromatography ◽  
Serum Proteins ◽  
Direct Injection ◽  
Functional Materials ◽  
Size Exclusion ◽  
Dna Nanostructure ◽  
Exclusion Chromatography ◽  
The Stability

DNA-based nanostructures (DNs) are advantageous for the design of functional materials for biology and medicine due to the nanoscale control provided by their predictable self-assembly. However, the use of DNs in vivo has been limited due to structural instability in biofluids. As the stability of a particular DN sets the scope of its potential biological applications, efficient methods to characterize stability are required. Here, we apply size exclusion chromatography (SEC) to study the stability of a tetrahedron DNA nanostructure (TDN) and demonstrate the analytical capabilities of our method in characterizing degradation by enzymes and a diluted human serum matrix. We show that SEC analysis can reliably assay TDN degradation by a nuclease through direct injection and peak integration. Furthermore, data analysis using a ratio chromatogram technique enables TDN peak deconvolution from the matrix of serum proteins. Using our method, we found that TDNs exhibit half-lives of 23.9 hours and 10.1 hours in 20% and 50% diluted human serum, respectively, which is consistent with reported stability studies in 10% fetal bovine serum. We anticipate that this method could be broadly applicable to characterize a variety of DNs and serve as an efficient technique toward analysis of the stability of new DN designs in complex biological matrixes.

scholarly journals Butyrylcholinesterase–Protein Interactions in Human Serum

2021 ◽  
Vol 22 (19) ◽  
pp. 10662
Author(s):  
Jacek Jasiecki ◽  
Anna Szczoczarz ◽  
Dominik Cysewski ◽  
Krzysztof Lewandowski ◽  
Piotr Skowron ◽  
...  
Keyword(s):  
Human Serum ◽  
Protein Interactions ◽  
Serum Proteins ◽  
Protein Complexes ◽  
Density Lipoprotein ◽  
Specific Protein ◽  
Size Exclusion ◽  
Strong Interactions ◽  
Binding Studies ◽  
Exclusion Chromatography

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein–protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes′ specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

scholarly journals Studies in Bisalbuminemia: Binding Properties of the Two Albumins

Blood ◽  
1962 ◽  
Vol 20 (2) ◽  
pp. 156-164 ◽  
Author(s):  
EDWARD J. SARCIONE ◽  
C. WILLIAM AUNGST
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Serum Protein ◽  
Serum Proteins ◽  
Protein Pattern ◽  
Total Serum ◽  
Binding Properties ◽  
Normal Human ◽  
Serum Components

Abstract 1. An abnormal serum protein pattern in a patient with Wegener’s granulomatosis and five of his relatives was identified as bisalbuminemia by electrophoretic and immunochemical methods. 2. With the exception of the patient with Wegener’s syndrome, the presence of bisalbuminemia was not associated with a significant change in total serum proteins, total albumin, serum components other than albumin, or any disease. 3. Addition of I131-thyroxine to bisalbumin sera resulted in thyroxine binding by albumin B but not by albumin A. The failure of albumin A to bind added I131-thyroxine leads to speculation that, in this family, neither albumin A nor B are identical to normal human serum albumin.

scholarly journals Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes.

1996 ◽  
Vol 16 (5) ◽  
pp. 2128-2134 ◽  
Author(s):  
J Papkoff ◽  
B Rubinfeld ◽  
B Schryver ◽  
P Polakis
Keyword(s):  
Signal Transduction Pathway ◽  
Size Exclusion ◽  
Beta Catenin ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Steady State Levels ◽  
Exogenous Expression ◽  
The Stability ◽  
Related Proteins ◽  
Cell Adhesion Proteins

The Wnt-1 proto-oncogene induces the accumulation of beta-catenin and plakoglobin, two related proteins that associate with and functionally modulate the cadherin cell adhesion proteins. Here we have investigated the effects of Wnt-1 expression on the tumor suppressor protein APC, which also associates with catenins. Expression of Wnt-1 in two different cell lines greatly increased the stability of APC-catenin complexes. The steady-state levels of both catenins and APC were elevated by Wnt-1, and the half-lives of both beta-catenin and plakoglobin associated with APC were also markedly increased. The stabilization of catenins by Wnt-1 was primarily the result of a selective increase in the amount of uncomplexed, monomeric beta-catenin and plakoglobin, detected both by affinity precipitation and size-exclusion chromatography of cell extracts. Exogenous expression of beta-catenin was possible in cells already responding to Wnt-1 but not in the parental cells, suggesting that Wnt-1 inhibits an essential regulatory mechanism for beta-catenin turnover. APC has the capacity to oppose this Wnt-1 effect in experiments in which overexpression of the central region of APC significantly reduced the size of the monomeric pool of beta-catenin induced by Wnt-1. Thus, the Wnt-1 signal transduction pathway leads to the accumulation of monomeric catenins and stabilization of catenin complex formation with both APC and cadherins.

scholarly journals Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp)

2021 ◽  
Vol 22 (15) ◽  
pp. 7777
Author(s):  
Lydia K. Muranova ◽  
Vladislav M. Shatov ◽  
Andrey V. Slushchev ◽  
Nikolai B. Gusev
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Quaternary Structure ◽  
Small Heat Shock Protein ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Wild Type ◽  
Simple Method ◽  
Exclusion Chromatography ◽  
Shock Proteins

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17–29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the β7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

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