title: Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling.
abstract: Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.
authors.affiliation: Department of Research Core Services, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Belinda
authors.initials: B
authors.lastname: Willard
authors.forename: Nadia
authors.initials: N
authors.lastname: Rachdaoui
authors.forename: John P
authors.initials: JP
authors.lastname: Kirwan
authors.forename: Rovshan G
authors.initials: RG
authors.lastname: Sadygov
authors.forename: William C
authors.initials: WC
authors.lastname: Stanley
authors.forename: Stephen
authors.initials: S
authors.lastname: Previs
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: McCullough
authors.forename: Takhar
authors.initials: T
authors.lastname: Kasumov
mesh_terms: D000818:Animals; D053299:Apolipoprotein B-100 / Q000097:blood*; D002853:Chromatography, Liquid; D017666:Deuterium Oxide / Q000378:metabolism*; D005215:Fasting / Q000097:blood; D005340:Fibrinogen / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D010455:Peptides / Q000032:analysis; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000032:analysis*; D012984:Software; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry; D014357:Trypsin
chemical_list: D053299:Apolipoprotein B-100; D010455:Peptides; D020543:Proteome; D012709:Serum Albumin; D005340:Fibrinogen; D014357:Trypsin; D017666:Deuterium Oxide