Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm.
We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.
Department of Gastroenterology, Cleveland Clinic, Cleveland, OH 44195, USA. kasumot@ccf.org
Takhar
T
Kasumov
Serguey
S
Ilchenko
Ling
L
Li
Nadia
N
Rachdaoui
Rovshan G
RG
Sadygov
Belinda
B
Willard
Arthur J
AJ
McCullough
Stephen
S
Previs
D000465:Algorithms; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D007553:Isotope Labeling; D010455:Peptides / Q000032:analysis; D014176:Protein Biosynthesis; D051381:Rats; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; D010455:Peptides; D012709:Serum Albumin; D003903:Deuterium
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