title: In vivo labeling: a glimpse of the dynamic proteome and additional constraints for protein identification.
abstract: Identities ascribed to the intact protein ions detected in MALDI-MS of whole bacterial cells or from other complex mixtures are often ambiguous. Isolation of candidate proteins can establish that they are of correct molecular mass and sufficiently abundant, but by itself is not definitive. An in vivo labeling strategy replacing methionine with selenomethionine has been employed to deliver an additional constraint for protein identification, i.e., number of methionine residues, derived from the shift in mass of labeled versus unlabeled proteins. By stressing a culture and simultaneously labeling, it was possible to specifically image the cells' response to the perturbation. Because labeled protein is only synthesized after application of the stress, it provides a means to view dynamic changes in the cellular proteome. These methods have been applied to identify a 15,879 Da protein ion from E. coli that was induced by an antibacterial agent with an unknown mechanism of action as SpY, a stress protein produced abundantly in spheroplasts. It has also allowed us to propose protein identities (and eliminate others from consideration) for many of the ions observed in MALDI (and ESI-MS) whole cell profiling at a specified growth condition.
authors.affiliation: Department of Biological Chemistry, University of California-Los Angeles, 90095-1570, USA. rloo@mednet.ucla.edu
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogorzalek Loo
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Ping
authors.initials: P
authors.lastname: Du
authors.forename: Tod
authors.initials: T
authors.lastname: Holler
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002138:Calibration; D016172:DNA Fingerprinting; D004926:Escherichia coli / Q000737:chemistry; D007525:Isoelectric Focusing; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000596:Amino Acids; D000900:Anti-Bacterial Agents; D011506:Proteins; D020543:Proteome