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
Site-specific methylation of Notch1 controls the amplitude and duration of the Notch1 response
Kerstin Hein,Kerstin Hein,Gerhard Mittler,Gerhard Mittler,Wiebke Cizelsky,Michael Kühl,Francesca Ferrante,Robert Liefke,Ina M. Berger,Steffen Just,J. Eric Sträng,Hans A. Kestler,Hans A. Kestler,Franz Oswald,Tilman Borggrefe,Tilman Borggrefe +15 more
TL;DR: It is found that the methyltransferase CARM1 methylated NICD at five conserved arginine residues within the C-terminal transactivation domain, which is important for its transcriptional activity and protein turnover.
Journal ArticleDOI
Detergent-Based but Gel-Free Method Allows Identification of Several Hundred Membrane Proteins in Single LC-MS Runs
TL;DR: A procedure that allows use of detergents and in-solution digestion of proteins and demonstrates that membrane proteins can be analyzed as efficiently as soluble proteins.
Journal ArticleDOI
ADF/Cofilin Regulates Secretory Cargo Sorting at the TGN via the Ca2+ ATPase SPCA1
Julia von Blume,Anne-Marie Alleaume,Gerard Cantero-Recasens,Amy J. Curwin,Amado Carreras-Sureda,Timo Zimmermann,Josse van Galen,Yuichi Wakana,Miguel A. Valverde,Vivek Malhotra +9 more
TL;DR: It is proposed that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca( 2+).
Journal ArticleDOI
SETD2 Restricts Prostate Cancer Metastasis by Integrating EZH2 and AMPK Signaling Pathways.
Huairui Yuan,Ying Han,Xuege Wang,Ni Li,Qiuli Liu,Yuye Yin,Hanling Wang,Lulu Pan,Li Li,Kun Song,Tong Qiu,Qiang Pan,Qilong Chen,Guoying Zhang,Yi Zang,Minjia Tan,Jian Zhang,Qintong Li,Xiaoming Wang,Jun Jiang,Jun Qin,Jun Qin +21 more
TL;DR: It is demonstrated that the SETD2-EZH2 axis integrates metabolic and epigenetic signaling to restrict PCa metastasis and it is identified that metformin-stimulated AMPK signaling converges at FOXO3 to stimulateSETD2 expression.
Journal ArticleDOI
Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale
Jing-Ming Dong,Felicia P. L. Tay,Hannah L. F. Swa,Jayantha Gunaratne,Jayantha Gunaratne,Thomas Leung,Brian Burke,Edward Manser,Edward Manser +8 more
TL;DR: The use of proximity-dependent biotinylation in U2OS osteosarcoma cells and stable isotope–labeled mass spectrometry to define macromolecular complexes enable the correct identification of therapeutic targets within the adhesome are illustrated.
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