"Out-gel" tryptic digestion procedure for chemical cross-linking studies with mass spectrometric detection

Journal of Proteome Research
Evgeniy V PetrotchenkoChristoph H Borchers

Abstract

SDS-PAGE is one of the most powerful protein separation techniques, and in-gel digestion is the leading method for converting proteins separated by SDS-PAGE into peptides suitable for mass spectrometry-based proteomic studies. In in-gel digestion, proteins are digested within the gel matrix, and the resulting peptides are extracted into an appropriate buffer. Transfer of the digested peptides to the liquid phase for subsequent mass spectrometric analysis, however, may be hampered by peptide-specific characteristics, including size, shape, poor solubility, adsorption to the polyacrylamide, and-in the case of cross-linking applications-by the branched structure of the peptides produced. This can be a limitation in cross-linking studies where efficient recoveries of the cross-linked peptides are critical. To overcome this limitation, we have developed a modification to the standard in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins, based on older passive diffusion methods. By omitting the gel staining and gel fixation steps, intact proteins or cross-linked protein complexes can move through the gel and into the buffer solution. Digestion of the entire protein in the buffer outside the gel increases the probab...Continue Reading

References

Jan 1, 1990·Methods in Enzymology·M G Harrington
Mar 1, 1996·Analytical Chemistry·A ShevchenkoM Mann
Dec 10, 1996·Proceedings of the National Academy of Sciences of the United States of America·A ShevchenkoM Mann
Oct 25, 2001·Analytical Biochemistry·J M Scheer, C A Ryan
Sep 7, 2004·Electrophoresis·Mohammad N SarboloukiHossain-Ali Rafiee-Pour
May 17, 2005·Methods : a Companion to Methods in Enzymology·Barbara KartenJean E Vance
Jun 15, 2005·Journal of Proteome Research·David R LoiselleChristoph H Borchers
Dec 8, 2005·Molecular & Cellular Proteomics : MCP·Leigh Anderson, Christie L Hunter
Apr 5, 2007·Nature Protocols·Andrej ShevchenkoMatthias Mann
Oct 7, 2008·Methods : a Companion to Methods in Enzymology·Paschalia M MountziarisAntonios G Mikos
Oct 8, 2008·Molecular & Cellular Proteomics : MCP·Evgeniy V PetrotchenkoChristoph H Borchers
May 1, 2009·Journal of Proteome Research·Merel A NessenChris G de Koster
Aug 1, 2009·Analytical Chemistry·Funing YanRuth Hogue Angeletti
Sep 15, 2009·Journal of the American Society for Mass Spectrometry·Ji Eun LeeNeil L Kelleher
Nov 7, 2009·Methods in Enzymology·Richard R Burgess
Jan 30, 2010·BMC Bioinformatics·Evgeniy V Petrotchenko, Christoph H Borchers
Jul 14, 2010·Molecular & Cellular Proteomics : MCP·Evgeniy V PetrotchenkoChristoph H Borchers
Jun 19, 2012·Journal of Mass Spectrometry : JMS·Diego Esteban-FernándezMichael W Linscheid

❮ Previous
Next ❯

Citations


❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.