A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude. Molecular & Cellular Proteomics 1:148 -156, 2002.
We have developed a method for rapidly cleaving and identifying proteins electroblotted onto poly(vinylidene difluoride) membranes. Cleavage is performed with 10% acetic acid in 7 M guanidine chloride at pH 2.5 for 1 h at 90 degrees C, resulting in fragmentation primarily at aspartyl-prolyl bonds. Peptides resulting from non-Asp-Pro cleavage are N-terminally blocked by reaction with orthophthalaldehyde (OPA) prior to automated Edman degradation. Reaction with OPA after cleavage blocks all amino acids containing primary amino groups. Only peptides containing an N-terminal amino acid with a secondary amino group (proline) will be available for reaction with the Edman reagent. The sequences obtained are used for protein database searching. Using this approach, proteins that are found to be N-terminally blocked can be removed from the sequencer, cleaved with acetic acid, blocked with OPA, and reapplied to the sequencer. The protein can then be identified from a database search using the sequence mixture obtained.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.