Journal ArticleDOI
In-gel digestion for mass spectrometric characterization of proteins and proteomes
Reads0
Chats0
TLDR
This protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.Abstract:
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.read more
Citations
More filters
Journal ArticleDOI
Analyzing protein-protein interactions by quantitative mass spectrometry.
TL;DR: Two detailed q-AP-MS protocols for pull-downs with immobilized bait proteins or transient transfection of tagged expression constructs are provided and critical parameters that need to be considered are discussed.
Journal ArticleDOI
Autophagy dysfunction and ubiquitin-positive protein aggregates in Dictyostelium cells lacking Vmp1.
TL;DR: The results suggest that Vmp1 is required for the clearance of these ubiquitinated protein aggregates through autophagy and highlight a potential role for VMP1 in protein-aggregation diseases.
Journal ArticleDOI
Quantitative Proteomic Analysis of Cellular Protein Modulation upon Inhibition of the NEDD8-Activating Enzyme by MLN4924
Hua Liao,Xiaozhen J. Liu,Jonathan L. Blank,David C. Bouck,Hugues Bernard,Khristofer Garcia,Eric S. Lightcap +6 more
TL;DR: The combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.
Journal ArticleDOI
Random protein sequences can form defined secondary structures and are well-tolerated in vivo.
Vyacheslav Tretyachenko,Vyacheslav Tretyachenko,Jiří Vymětal,Jiří Vymětal,Lucie Bednárová,Vladimír Kopecký,Kateřina Hofbauerová,Helena Jindrová,Helena Jindrová,Martin Hubálek,Radko Souček,Jan Konvalinka,Jan Konvalinka,Jiří Vondrášek,Klára Hlouchová,Klára Hlouchová +15 more
TL;DR: It is found that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.
Journal ArticleDOI
Toll-like receptor mediated inflammation requires FASN-dependent MYD88 palmitoylation.
Young Chan Kim,Sang Eun Lee,Somi K. Kim,Hyun Duk Jang,Injoo Hwang,Sooryeonhwa Jin,Sooryeonhwa Jin,Eun Byeol Hong,Kyoung-Soon Jang,Hyo-Soo Kim +9 more
TL;DR: Inhibition of fatty acid synthase by C75 improved neutrophil chemotaxis and increased the survival of mice with sepsis in cecal ligation puncture and lipopolysaccharide-induced septic shock models, suggesting intracellular saturated fatty acid-dependent palmitoylation of MYD88 by ZDHHC6 is a therapeutic target of sepsi.
References
More filters
Journal ArticleDOI
Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels
TL;DR: Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining, and this work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
Journal ArticleDOI
Mass spectrometry-based proteomics
Ruedi Aebersold,Matthias Mann +1 more
TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Journal ArticleDOI
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
Shao En Ong,Blagoy Blagoev,Irina Kratchmarova,Dan B. Kristensen,Hanno Steen,Akhilesh Pandey,Matthias Mann +6 more
TL;DR: SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system and is applied to the relative quantitation of changes in protein expression during the process of muscle cell differentiation.
Journal ArticleDOI
Stop and Go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and lc/ms sample pretreatment in proteomics
TL;DR: A novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips, finding that the Stage system is well-suited as a universal sample preparation system for proteomics.
Journal ArticleDOI
Mass spectrometry-based proteomics turns quantitative.
Shao En Ong,Matthias Mann +1 more
TL;DR: Two recently developed methodologies offer the opportunity to obtain quantitative proteomic information by comparing the signals from the same peptide under different conditions, and stable isotope labels facilitates direct quantification from the mass spectra.
Related Papers (5)
MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
Jiirgen Cox,Matthias Mann +1 more