scholarly journals Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Kerrie A. Morrison ◽  
Kate J. Heesom ◽  
Karen J. Edler ◽  
James Doutch ◽  
Gareth J. Price ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Mammalian Cells ◽  
Analytical Techniques ◽  
Protein Characterization ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Detergent Extraction

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

scholarly journals Identification of cytoplasmic sialidase NEU2-associated proteins by LC-MS/MS

2018 ◽  
Vol 44 (4) ◽  
pp. 462-472
Author(s):  
Secil Akyildiz Demir ◽  
Volkan Seyrantepe
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Actin Filaments ◽  
Western Blot Analysis ◽  
Mammalian Cells ◽  
Protein S ◽  
Mass Spectrometry Analysis ◽  
Interacting Protein ◽  
Spectrometry Analysis

Abstract Background Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, glycopeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results Here, mass spectrometry analysis showed four proteins; α-actin, β-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.

scholarly journals Determination of the lipid composition of the GPI anchor

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256184
Author(s):  
Auxiliadora Aguilera-Romero ◽  
Susana Sabido-Bozo ◽  
Sergio Lopez ◽  
Alejandro Cortes-Gomez ◽  
Sofia Rodriguez-Gallardo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Composition ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Gpi Anchor ◽  
Spectrometry Analysis ◽  
Lipid Structure ◽  
Lipid Moiety ◽  
Protein Lipidation

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.

scholarly journals Miniaturized Digestion and Extraction of Surface Proteins from Candida albicans following Treatment with Histatin 5 for Mass Spectrometry Analysis

2016 ◽  
Vol 2016 ◽  
pp. 1-12
Author(s):  
Shirley Fan ◽  
Eduardo B. Moffa ◽  
Yizhi Xiao ◽  
Walter L. Siqueira ◽  
Ken K.-C. Yeung
Keyword(s):  
Mass Spectrometry ◽  
Candida Albicans ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Bacterial Cells ◽  
Intact Cells ◽  
Histatin 5 ◽  
Spectrometry Analysis ◽  
Glass Slides ◽  
Glass Surfaces

A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at μL volumes. Specifically, Candida albicans cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.

scholarly journals Quantitative Proteomic Analysis of Biogenesis-Based Classification for Extracellular Vesicles

Proteomes ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 33
Author(s):  
Linwen Zhang ◽  
Jeremie Parot ◽  
Vincent A. Hackley ◽  
Illarion V. Turko
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Specific Protein ◽  
Mass Spectrometry Analysis ◽  
Protein Markers ◽  
Spectrometry Analysis ◽  
Endosomal Membrane ◽  
Size Limitation ◽  
Endosomal Membranes

Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.

scholarly journals Extraction, Isolation and Identification of Antimicrobial Substances from Bacillus amyloliquefaciens CMN1308

2017 ◽  
Vol 45 (1) ◽  
pp. 308-315
Author(s):  
Yingyou FANG ◽  
Linling LI ◽  
Yongliang ZHENG ◽  
Honghui YUAN ◽  
Xuehua ZHANG ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Bacillus Amyloliquefaciens ◽  
Protein Characterization ◽  
Mass Spectrometry Analysis ◽  
Antimicrobial Protein ◽  
Isolation And Identification ◽  
Spectrometry Analysis ◽  
Antimicrobial Substances ◽  
Sds Page

Four separation methods of antimicrobial substances produced by CMN1308 (Bacillus amyloliquefaciens) were evaluated and selected according to number of antimicrobial substances and its activity in vitro. The results showed that extraction by acid precipitation of the fermentation supernatant of CMN1308 was the best with a diameter of inhibition zone of pathogen fungi P. expansum of 12.3 mm in a laboratory bioassay. Applying a silica thin layer chromatography (TLC), SDS-PAGE and other separation technologies we isolate antimicrobial substances, and the separated band were cut off for mass spectrometry analysis. The TLC of crude extract of CMN1308 show a topical band corresponding with the surfactin standard (Rf value =0.75), proved that the strain CMN1308 can produce this surface active compound. The mycoprotein extracted from CMN1308 was separated by Tricine-SDS-PAGE modified with the addition of urea in the separation gel. After mass spectrometric analysis and protein characterization, the isolated mycoprotein showed a maximum ion peak at M/Z of 2679 and molecular weight of 29.5 kDa, matching with protein flagellin. The extracellular antimicrobial protein of strain CMN1308 display four bands after urea-Tricine-SDS-PAGE, but after mass spectrometry analysis only two bands were identified. Band “A” with a maximum ion peak at M/Z of 1926 and molecular weight of 49.8 kDa, aligned with NCBI database, matching with DLDH (dihydrolipoamide dehydrogenase enzyme). Band “D” show the maximum ion peak at M/Z of 2936 and molecular weight of 22.4 kD, matching with a chitin binding protein. Thus, the strain CMN1308 has the potential to be developed as a commercial biological control agent for chestnut common pathogenic fungi.

MALDI-TOF Mass Spectrometry Analysis of Amphipol-Trapped Membrane Proteins

2012 ◽  
Vol 84 (14) ◽  
pp. 6128-6135 ◽  
Author(s):  
Chérine Bechara ◽  
Gérard Bolbach ◽  
Paola Bazzaco ◽  
K. Shivaji Sharma ◽  
Grégory Durand ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof

scholarly journals DART-ID increases single-cell proteome coverage

2018 ◽  
Author(s):  
Albert T. Chen ◽  
Alexander Franks ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Single Cell ◽  
Retention Time ◽  
Mammalian Cells ◽  
Statistical Power ◽  
Single Cells ◽  
Peptide Sequence ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis

AbstractAnalysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS) can identify and quantify thousands of proteins in microgram-level samples, such as those comprised of thousands of cells. This process, however, remains challenging for smaller samples, such as the proteomes of single mammalian cells, because reduced protein levels reduce the number of confidently sequenced peptides. To alleviate this reduction, we developed Data-driven Alignment of Retention Times for IDentification (DART-ID). DART-ID implements principled Bayesian frameworks for global retention time (RT) alignment and for incorporating RT estimates towards improved confidence estimates of peptide-spectrum-matches. When applied to bulk or to single-cell samples, DART-ID increased the number of data points by 30 – 50% at 1% FDR, and thus decreased missing data. Benchmarks indicate excellent quantification of peptides upgraded by DART-ID and support their utility for quantitative analysis, such as identifying cell types and cell-type specific proteins. The additional datapoints provided by DART-ID boost the statistical power and double the number of proteins identified as differentially abundant in monocytes and T-cells. DART-ID can be applied to diverse experimental designs and is freely available at http://github.com/SlavovLab/DART-ID.Author SummaryIdentifying and quantifying proteins in single cells gives researchers the ability to tackle complex biological problems that involve single cell heterogeneity, such as the treatment of solid tumors. Mass spectrometry analysis of peptides can identify their sequence from their masses and the masses of their fragment ion, but often times these pieces of evidence are insufficient for a confident peptide identification. This problem is exacerbated when analyzing lowly abundant samples such as single cells. To identify even peptides with weak mass spectra, DART-ID incorporates their retention time – the time when they elute from the liquid chromatography used to physically separate them. We present both a novel method of aligning the retention times of peptides across experiments, as well as a rigorous framework for using the estimated retention times to enhance peptide sequence identification. Incorporating the retention time as additional evidence leads to a substantial increase in the number of samples in which proteins are confidently identified and quantified.

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