Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry

Abstract

Mirza SP, Olivier M. Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry.

Quantitative characterization of changes in the cardiac mitochondrial proteome during anesthetic preconditioning and ischemia

“…18 O (Cambridge Isotope Laboratories, Andover, MA) according to previously published protocols (12,20). Briefly, precipitated proteins for each treatment were divided equally and resuspended in 250 mM ammonium bicarbonate made with either H 2 16 O or H2 18 O.…”

“…The digests were then incubated at 37°C for another 12 h. Digestions were stopped with the addition of 1 l 1% (vol/vol) formic acid. Alternatively, samples were digested on trypsin spin columns (20). Prior to being desalted on modified C 18 Zip-Tips (Millipore, Billerica, MA), samples were mixed 1:1 according to the diagram illustrated in Fig.…”

“…On the basis of these initial findings, we aimed in this study to quantitatively analyze changes in protein levels specific to cardiac mitochondria not only after exposure to the preconditioning agent but after exposure to ischemia and reperfusion as well. Quantitative proteomic approaches using isotopic labeling and mass spectrometry allowed for the relative comparison of protein abundances in cardiac mitochondrial samples from control and isoflurane exposed rats before and after ischemia (12,20).…”

Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat

“…Mass spectral analysis was carried out on a ThermoFinnigan LTQ ion trap mass spectrometer interfaced with a Surveyor LC system in the Proteomics Center of the MCW Biotechnology and Bioengineering Center (http://proteomics. mcw.edu) as described previously (36,48,49) with some modifications. Delipidated samples were resuspended in 250 mM ammonium bicarbonate (ABC) solution, reduced with 10 mM dithiothreitol (DTT) for 30 min at 37°C, cysteines were alkylated with 55 mM iodoacetamide for 45 min at 37°C (in the dark), and then proteins digested overnight at 37°C with trypsin (ratio 1:50).…”

Variability of healthy human proteome

“…В настоящее время существует только несколько работ, касающихся этого вопроса [29][30][31][32][33]. Возможно, недостаточность литературных данных о многообразии посттрансляционных модификаций белков отчасти объясняется тем, что отдельные их типы не только обратимы, но и короткоживущие, что методически крайне затрудняет их выявление [34][35][36].…”