NewS

By Chia-Feng Tsai & Yasushi Ishihama, Kyoto University

Dysregulation of cellular signaling based on protein phosphorylation is closely linked to pathogenesis of human diseases and therapeutic strategies to control the phospho-signaling have been accepted to develop molecular-targeting drugs for cancer. MS-based quantitative phosphoproteomic approaches have been widely used to quantify over 10,000 phosphorylation sites by utilizing strong cation exchange chromatography, hydrophilic interaction chromatography or basic-pH reversed-phase chromatography to fractionate the complex samples. However, such extensive fractionation approaches require longer LC-MS measurement time as well as tedious pretreatment steps, resulting in reduced throughput and low reproducibility. Besides, these approaches cannot be applied to primary cells of rare tissues or clinical biopsy samples due to the limited starting materials.

To address these issues, Humphrey et al. (from Matthias Mann’s group) at Max Planck Institute of Biochemistry have developed a streamlined phosphoproteomics workflow called “EasyPhos” which has been designed as a high throughput and simplified workflow to study time-resolved phosphorylation alteration in vivo without any pre-fractionation strategy. They used trifluoroethanol for the digestion buffer which allows bypassing the peptide desalting step before phosphopeptide enrichment. This protocol can reduce the potential sample loss during the desalting process implemented in the conventional protocols. Besides, this simplified procedure can be expanded to a 96-well plate format to increase the throughput of phosphopeptide enrichment. The combination of this workflow with Q Exactive benchtop Orbitrap mass spectrometer allows monitoring of more than 10,000 phosphorylation sites from a mouse cell line by single-shot LC-MS/MS analysis with 2hr gradient. The applicability of this parallelized EasyPhos workflow has been demonstrated on the analysis of liver phosphoproteomes at different time points (early and intermediate) in fasted mice under insulin exposure. Up to 31,605 phosphopeptides (25,507 phosphorylation sites belong to class 1) from 6,138 phosphoproteins were identified from 91 biologically distinct liver tissues by the high throughput 96-well EasyPhos assay. Importantly, at least six biological replicate analyses (separate mice) per sample for each time point provide a highly statistical power to illustrate time-resolved maps of insulin signaling. Moreover, these dynamics datasets illuminate not only the insulin-mediated signaling network but also the signaling cascade from cell surface to the nucleus within 1 min in vivo. This rapid and high throughput EasyPhos workflow will facilitate to accumulate the knowledges of cellular signaling dynamics under physiological or pathological regulation.

Figure reprinted with permission of the Nature Publishing Group

This study was reported in the journal of Nature Biotechnology on August 17, 2015.

Link: http://www.nature.com/nbt/journal/v33/n9/abs/nbt.3327.html

Read the full article here: Prioritizing Proteomics Assay Development for Clinical Translation

Vol. 66, No. 2, 2015
Journal of the American College of Cardiology
© 2015 by the American College of Cardiology Foundation
Published by Elsevier Inc.

Richard D. Semba, Maggie Lam, Kai Sun, Pingbo Zhang, Debra A. Schaumberg, Luigi Ferrucci, Peipei Ping, and Jennifer E. Van Eyk

Read the full article here: Priorities and trends in the study of proteins in eye research, 1924-2014

www.clinical.proteomics-journal.com

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics Clin. Appl. 2015, 00, 1-18

DOI 10.1002/prca.201500006

By Neil Savage

The effort to catalogue proteins goes deeper in a push to make genetics research deliver practical benefits. 

Published in Nature: S6 | NATURE | VOL 527 | NOV 2015

For more background, read the Metrics for the Human Proteome Project 2015.

Read the full article here: State of the Human Proteome in 2014/2015 As Viewed through PeptideAtlas

Read the full article here: JPR-Metrics for the Human Proteome Project 2015

Niphakis, M.J. et al. Cell 161, 1668–1680 (18 June 2015)
News in Science (HUPOST Vol. 5, Q3 September 2015)

Lipids are a class of hydrocarbon-containing natural molecules; important examples include some vitamins (A, D, E), hormones (estrogen, testosterone), neurotransmitters (endocannabinoids) and components of fat (triglycerides, cholesterol). Their biological functions to regulate cell physiology and disease are often mediated through interactions with proteins. Despite the fact that a substantial number of drug targets are lipid-binding proteins, it has been challenging to establish a global portrait of how lipids interact with proteins in cells.

Benjamin F. Cravatt’s team at The Scripps Research Institute (TSRI) has devised a powerful chemical proteomic method to map the human lipid-binding proteome. In this study, Niphakis and colleagues described a set of fatty acid–based chemical probes combined with quantitative mass spectrometry to provide the first global portrait of lipid-protein interactions in human cells. Their results revealed a surprisingly large number of lipid-protein interactions from diverse functional classes, including some interactions which are already targeted by existing drugs and, most importantly, many novel lipid-binding proteins. The probes can also be used to identify the targets of drugs that disrupt lipid-protein interactions in cells, pointing to the broad potential of lipid-binding proteins for ligand/drug development.

“Traditionally, scientists have theorized fairly narrow limits for the types of proteins that can be targeted with small-molecule ligands, but our results suggest that those boundaries are much wider,” said Cravatt (adapted from News Release of The Scripps Research Institute)

This study was reported in the journal of Cell on June 18, 2015.

Figure provided by Benjamin F. Cravatt