title: Measuring H(2)(18)O tracer incorporation on a QQQ-MS platform provides a rapid, transferable screening tool for relative protein synthesis.
abstract: Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H(2)(18)O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H(2)(18)O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment.
authors.affiliation: Merck Research Laboratories , 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States.
authors.forename: James P
authors.initials: JP
authors.lastname: Conway
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Johns
authors.forename: Sheng-Ping
authors.initials: SP
authors.lastname: Wang
authors.forename: Nykia D
authors.initials: ND
authors.lastname: Walker
authors.forename: Thomas A
authors.initials: TA
authors.lastname: McAvoy
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhou
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhao
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Previs
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Roddy
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hubbard
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Yates
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Hendrickson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053299:Apolipoprotein B-100 / Q000096:biosynthesis / Q000302:isolation & purification; D004285:Dogs; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008077:Lipoproteins, LDL / Q000097:blood / Q000302:isolation & purification; D008079:Lipoproteins, VLDL / Q000097:blood / Q000302:isolation & purification; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009842:Oligopeptides / Q000737:chemistry; D010103:Oxygen Isotopes; D014176:Protein Biosynthesis; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D053299:Apolipoprotein B-100; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D009842:Oligopeptides; D010103:Oxygen Isotopes