scholarly journals A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

2019 ◽  
Author(s):  
K. Adam ◽  
S. Fuhs ◽  
J. Meisenhelder ◽  
A. Aslanian ◽  
J. Diedrich ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibodies ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
New Era ◽  
Spectrometry Analysis ◽  
Immunoaffinity Purification ◽  
Long Time ◽  
Living Organisms ◽  
Histidine Phosphorylation

AbstractFour types of phosphate-protein linkage generate nine different phosphoresidues in living organisms. Histidine phosphorylation is a long-time established but largely unexplored post-translational modification, mainly because of the acid-lability of the phosphoramidate bonds. This lability means that standard phosphoproteomic methods used for conventional phosphate esters (phospho-Ser/Thr/Tyr) must be modified to analyze proteins containing the phosphoramidate-amino acids - phospho-His/Arg/Lys. We show that a non-acidic method allows enrichment of non-conventional phosphoresidue-containing peptides from tryptic digests of human cell lines, using hydroxyapatite binding and/or immobilized 1-pHis and 3-pHis monoclonal antibodies for enrichment. 425 unique non-conventional phosphorylation sites (i.e. pHis, pLys and pArg) were detected with a high probability of localization by LC-MS/MS analysis and identified using a customized MaxQuant configuration, contributing to a new era of study in post-translational modification and cell signaling in humans. This is the first fully non-acidic method for phosphopeptide enrichment which uses immunoaffinity purification and remains compatible with mass spectrometry analysis for a wider coverage of potential protein phosphorylation events.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

scholarly journals Exploring the Lipidome: Current Lipid Extraction Techniques for Mass Spectrometry Analysis

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 231 ◽  
Author(s):  
Julian Aldana ◽  
Adriana Romero-Otero ◽  
Mónica P. Cala
Keyword(s):  
Mass Spectrometry ◽  
Energy Homeostasis ◽  
Lipid Extraction ◽  
Structural Diversity ◽  
Extraction Yield ◽  
Mass Spectrometry Analysis ◽  
Extraction Techniques ◽  
Lipid Profiling ◽  
Spectrometry Analysis ◽  
Living Organisms

In recent years, high-throughput lipid profiling has contributed to understand the biological, physiological and pathological roles of lipids in living organisms. Across all kingdoms of life, important cell and systemic processes are mediated by lipids including compartmentalization, signaling and energy homeostasis. Despite important advances in liquid chromatography and mass spectrometry, sample extraction procedures remain a bottleneck in lipidomic studies, since the wide structural diversity of lipids imposes a constrain in the type and amount of lipids extracted. Differences in extraction yield across lipid classes can induce a bias on down-stream analysis and outcomes. This review aims to summarize current lipid extraction techniques used for untargeted and targeted studies based on mass spectrometry. Considerations, applications, and limitations of these techniques are discussed when used to extract lipids in complex biological matrices, such as tissues, biofluids, foods, and microorganisms.

Diazo Reagent Labeling with Mass Spectrometry Analysis for Sensitive Determination of Ribonucleotides in Living Organisms

2019 ◽  
Vol 92 (2) ◽  
pp. 2301-2309 ◽  
Author(s):  
Fei-Long Liu ◽  
Chu-Bo Qi ◽  
Qing-Yun Cheng ◽  
Jiang-Hui Ding ◽  
Bi-Feng Yuan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Sensitive Determination ◽  
Living Organisms
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