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
An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture
Sean C. Bendall,Sean C. Bendall,Christopher S. Hughes,J. Larry Campbell,Morag Stewart,Paula Pittock,Suya Liu,Eric Bonneil,Pierre Thibault,Mickie Bhatia,Gilles A. Lajoie +10 more
TL;DR: The IE-MS approach provides a simple and reliable technique that greatly enhances instrument performance by increasing the effective depth of MS-based proteomic profiling, and should be widely applicable to any LC-MS/MS instrument platform or biological system.
Journal ArticleDOI
Cyanobacterial reuse of extracellular organic carbon in microbial mats
Rhona K. Stuart,Xavier Mayali,Jackson Z. Lee,R. Craig Everroad,Mona Hwang,Brad M. Bebout,Peter K. Weber,Jennifer Pett-Ridge,Michael P. Thelen +8 more
TL;DR: Results indicate cyanobacteria reuse excess organic carbon, constituting a dynamic pool of extracellular resources in these mats, and demonstrate rapid incorporation ofextracellular-derived carbon.
Journal ArticleDOI
Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and Phosphoproteomics
TL;DR: In this article, the authors applied quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component, namely, the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes.
Journal ArticleDOI
Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors
TL;DR: The phosphoproteome of the mouse liver cell line Hepa1‐6 is investigated by stable isotope labeling by amino acids in cell culture (SILAC) and high resolution MS to derive an upper bound on phosphorylation occupancy.
Journal ArticleDOI
Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome
TL;DR: SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins.
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