title: Protein turnover quantification in a multilabeling approach: from data calculation to evaluation.
abstract: Liquid chromatography coupled to tandem mass spectrometry in combination with stable-isotope labeling is an established and widely spread method to measure gene expression on the protein level. However, it is often not considered that two opposing processes are responsible for the amount of a protein in a cell--the synthesis as well as the degradation. With this work, we provide an integrative, high-throughput method--from the experimental setup to the bioinformatics analysis--to measure synthesis and degradation rates of an organism's proteome. Applicability of the approach is demonstrated with an investigation of heat shock response, a well-understood regulatory mechanism in bacteria, on the biotechnologically relevant Corynebacterium glutamicum. Utilizing a multilabeling approach using both heavy stable nitrogen as well as carbon isotopes cells are metabolically labeled in a pulse-chase experiment to trace the labels' incorporation in newly synthesized proteins and its loss during protein degradation. Our work aims not only at the calculation of protein turnover rates but also at their statistical evaluation, including variance and hierarchical cluster analysis using the rich internet application QuPE.
authors.affiliation: Department of Plant Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany. christian.troetschel@rub.de
authors.forename: Christian
authors.initials: C
authors.lastname: Trötschel
authors.forename: Stefan P
authors.initials: SP
authors.lastname: Albaum
authors.forename: Daniel
authors.initials: D
authors.lastname: Wolff
authors.forename: Simon
authors.initials: S
authors.lastname: Schröder
authors.forename: Alexander
authors.initials: A
authors.lastname: Goesmann
authors.forename: Tim W
authors.initials: TW
authors.lastname: Nattkemper
authors.forename: Ansgar
authors.initials: A
authors.lastname: Poetsch
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000145:classification / Q000378:metabolism*; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000254:growth & development / Q000378:metabolism*; D018869:Heat-Shock Response; D006358:Hot Temperature; D020407:Internet; D007553:Isotope Labeling / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010455:Peptides