Proteome Dynamics with Heavy Water — Instrumentations, Data Analysis, and Biological Applications

Abstract

The quantitative assessment of the synthesis of individual proteins has been greatly hindered by the lack of a high-throughput nonradioactive method. We recently developed a method that we call proteome dynamics and software that enables high-throughput kinetic analyses of peptides on a proteome-wide scale. Previous studies established that oral administration of heavy water H O or deuterium oxide, D O is safe and well tolerated in humans. "riefly, a loading dose of H O, a nonradioactive isotope, is administered in drinking water. H O rapidly labels body water and transfers H from H O to H-labeled amino acids, which incorporates into proteins dependent upon the rate of synthesis of the specific protein. Proteins are analyzed by high-resolution mass spectrometry and protein synthesis is calculated using specialized software. We have established the effectiveness of this method for plasma and mitochondrial proteins. We demonstrated that fasting has a differential effect on the synthesis rates of proteins. We also applied this method to assess the effect of heart failure on the stability of mitochondrial proteins. In this review, we describe the study design, instrumentation, data analysis, and biological application of heavy water-based proteome turnover studies. We summarize this chapter with the challenges in the field and future directions.

Deuterium‐ und tritiummarkierte Verbindungen: Anwendungen in den modernen Biowissenschaften

“…Kürzlich wurden auch experimentelle Protokolle zur Messung der Zellproliferation basierend auf dem Deuteriumeinbau aus D 2 Oinden Deoxyribose-Baustein von Purin-Deoxyribunukleotiden in der DNAt eilender Zellen beschrieben. [224] Angewandte Chemie Aufsätze Das SILK-Protokoll ("stable isotope labeled kinetic") ist ein Ansatz zur Untersuchung der nicht-natürlichen Anreicherung amyloider Plaques,d ie sich in den Gehirnen von Alzheimer-Patienten finden, sowie der zugehçrigen Wirkung von Alzheimermedikamenten, wobei die Produktions-und Abbauraten von b-Amyloidpeptiden nach der Verabreichung von [ 13 C]l-Leucin gemessen werden. [6,6-D 2 ]-d-Glukose wurde beispielsweise eingesetzt, um die hepatische Glukoseproduktionsrate zu messen [230] oder die Insulinsensitivitätu nd Glukoseeffektivitätb ei basalem Insulin im intravençsen Glukosetoleranztest (IVGTT) zu bestimmen.…”

“…Transaminierungsprozess zur Deuteriummarkierung von Proteinend urch D 2 O. [224] Angewandte Chemie Aufsätze 1786 www.angewandte.de Das SILK-Protokoll ("stable isotope labeled kinetic") ist ein Ansatz zur Untersuchung der nicht-natürlichen Anreicherung amyloider Plaques,d ie sich in den Gehirnen von Alzheimer-Patienten finden, sowie der zugehçrigen Wirkung von Alzheimermedikamenten, wobei die Produktions-und Abbauraten von b-Amyloidpeptiden nach der Verabreichung von [ 13 C]l-Leucin gemessen werden. [232] Ein duales SILK-Protokoll, bestehend aus der oralen Gabe von Deuteriumoxid zusammen mit der konstanten Infusion von Natrium[1-13 C]acetat, wurde an zwçlf Te stpersonen angewendet, um die anteiligen und absoluten Syntheseraten von Cholesterin zu messen.…”

The C21-formyl group in chlorophyll f originates from molecular oxygen

“…Stable isotopes have been used successfully to determine the biosynthetic pathways of different molecules. Of these, 13 C, 15 N, and 18 O have been applied to understand the mechanisms of bacteriochlorophyll and Chl biosynthesis (6,10,11). The 18 O in H 2 18 O and 18 O 2 has been used to investigate the origin of the oxygen atoms in Chl a, b, and d (12)(13)(14).…”

“…The fifth oxygen atom at the C13 1 -oxo group of Chl a comes from molecular oxygen (O 2 ) through an oxygenase-type reaction in all oxygenic photosynthetic organisms (6). Using 18 O-labeling technology, it was further revealed that the sixth oxygen atom at the C7 formyl group of Chl b is derived from O 2 and not from water (12). The sixth oxygen atom of the formyl group at the C3 position in Chl d is also derived from O 2 through an oxygenase-type reaction mechanism, although the enzyme directly involved in the reaction is yet to be characterized (14).…”

“…Using 18 O labeling, we explored whether the origin of the oxygen atoms in Chl f was from water (H 2 18 O) or from oxygen ( 18 O 2 ). The kinetics of Chl a and Chl f biosynthesis in response to changed light conditions was investigated using D 2 O-seawater in the culture medium.…”