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 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 Primary Structure and Antibacterial Activity of Chicken Bone Marrow-Derived β-Defensins

2009 ◽  
Vol 53 (11) ◽  
pp. 4647-4655 ◽  
Author(s):  
Chrystelle Derache ◽  
Valérie Labas ◽  
Vincent Aucagne ◽  
Hervé Meudal ◽  
Céline Landon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bone Marrow ◽  
Posttranslational Modifications ◽  
Amino Acid Sequences ◽  
Biologically Active ◽  
Mass Spectrometry Analysis ◽  
Gram Positive ◽  
Intact Cells ◽  
Spectrometry Analysis ◽  
Chicken Bone

ABSTRACTThree biologically active β-defensins were purified by chromatography from chicken bone marrow extract: avian β-defensin 1 (AvBD1), AvBD2, and the newly isolated β-defensin AvBD7. Mass spectrometry analyses showed that bone marrow-derived AvBD1, -2, and -7 peptides were present as mature peptides and revealed posttranslational modifications for AvBD1 and AvBD7 in comparison to their in silico-predicted amino acid sequences. Tandem mass spectrometry analysis using the nanoelectrospray-quadrupole time of flight method showed N-terminal glutaminyl cyclization of mature AvBD7 and C-terminal amidation of mature AvBD1 peptide, while posttranslational modifications were absent in bone marrow-derived mature AvBD2 peptide. Furthermore, mass spectrometry analysis performed on intact cells confirmed the presence of these three peptides in mature heterophils. In addition, the antibacterial activities of the three β-defensins against a large panel of gram-positive and -negative bacteria were assessed. While the three defensins displayed similar antibacterial spectra of activity against gram-positive strains, AvBD1 and AvBD7 exhibited the strongest activity against gram-negative strains in comparison to AvBD2.

scholarly journals Online capillary electrophoresis – mass spectrometry analysis of histatin-5 and its degradation products

The Analyst ◽  
2020 ◽  
Vol 145 (14) ◽  
pp. 4787-4794
Author(s):  
Jared Lamp ◽  
Svetlana P. Ikonomova ◽  
Amy J. Karlsson ◽  
Qiangwei Xia ◽  
Yan Wang
Keyword(s):  
Mass Spectrometry ◽  
Capillary Electrophoresis ◽  
Degradation Products ◽  
Mass Spectrometry Analysis ◽  
Histatin 5 ◽  
Spectrometry Analysis ◽  
Salivary Peptide ◽  
Antibacterial And Antifungal Activities ◽  
Antibacterial And Antifungal ◽  
Capillary Electrophoresis Mass Spectrometry

Histatin-5 (Hst-5) is a human salivary peptide with antibacterial and antifungal activities. A novel capillary electrophoresis – mass spectrometry (CE-MS) method is developed to address issues related to highly basic and cationic nature of Hst-5.

scholarly journals Formation of carbohydrate-functionalised polystyrene and glass slides and their analysis by MALDI-TOF MS

2012 ◽  
Vol 8 ◽  
pp. 753-762 ◽  
Author(s):  
Martin J Weissenborn ◽  
Johannes W Wehner ◽  
Christopher J Gray ◽  
Robert Šardzík ◽  
Claire E Eyers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometric ◽  
Mass Spectrometry Analysis ◽  
Spectrometric Analysis ◽  
Spectrometry Analysis ◽  
Maldi Tof ◽  
Glass Slides ◽  
Array Format ◽  
Tof Ms

Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic, noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such ligands.

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.

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