scholarly journals Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

2020 ◽  
Author(s):  
Friedel Drepper ◽  
Jacek Biernat ◽  
Senthillvelrajan Kaniyappan ◽  
Helmut E. Meyer ◽  
Eva Maria Mandelkow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Native Mass Spectrometry ◽  
Eukaryotic Cell ◽  
Cell System ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis ◽  
Phosphorylated Peptides ◽  
Tau Aggregation

AbstractAbnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ∼20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi (± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi (± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.

scholarly journals Data on peptides identified by mass spectrometry analysis of in vitro DYRK1A-mediated phosphorylation sites on GLI1

Data in Brief ◽  
2017 ◽  
Vol 15 ◽  
pp. 577-583 ◽  
Author(s):  
Ben K. Ehe ◽  
David R. Lamson ◽  
Michael Tarpley ◽  
Rob U. Onyenwoke ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis

scholarly journals Ubiquitination of the yeast receptor Atg32 modulates mitophagy

2019 ◽  
Author(s):  
Pierre Vigié ◽  
Cécile Gonzalez ◽  
Stephen Manon ◽  
Ingrid Bhatia-Kissova ◽  
Nadine Camougrand
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Stationary Phase ◽  
Proteolytic Activity ◽  
Posttranslational Modifications ◽  
Protein Level ◽  
Mitochondrial Membrane ◽  
Mass Spectrometry Analysis ◽  
Outer Mitochondrial Membrane ◽  
Spectrometry Analysis

AbstractMitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of these organelles. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Some posttranslational modifications, such as phosphorylation, are crucial for the execution of the mitophagy process. In our study, we showed that in the stationary phase of growth, and to a lesser extent during starvation, the Atg32 protein level decreases. The fact that a decline in Atg32 level can be prevented by inhibition of the proteolytic activity of proteasome may indicate that Atg32 is also ubiquitylated. In fact, mass spectrometry analysis of purified Atg32 protein showed ubiquitination of lysine residue in position 282. These different patterns of posttranslational modifications of Atg32 could allow cells to control the mitophagy process carefully.

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 Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2134
Author(s):  
Barbara Deracinois ◽  
Aurélie Matéos ◽  
Audrey Romelard ◽  
Audrey Boulier ◽  
Julie Auger ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Hydrolysate ◽  
Casein Hydrolysate ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis ◽  
Casein Phosphopeptides ◽  
Liquid Chromatography Tandem Mass ◽  
Amino Acid Length ◽  
One Step

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

scholarly journals Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus

2021 ◽  
Vol 118 (42) ◽  
pp. e2108856118
Author(s):  
Claire M. Grison ◽  
Paul Lambey ◽  
Sylvain Jeannot ◽  
Elise Del Nero ◽  
Simon Fontanel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Staphylococcus Aureus ◽  
G Protein ◽  
Protein Complexes ◽  
Native Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Exchange Mass Spectrometry ◽  
Toxin Binding ◽  
And Function

Atypical chemokine receptor 1 (ACKR1) is a G protein–coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein–binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1−Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

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