Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

doi:10.1007/978-1-59745-188-8_21

Cited by 35 publications

(31 citation statements)

References 12 publications

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“…After destaining the Coomassie blue stained gel, 18 gel slides from high to low molecular weight were excised and cut into small pieces of no larger than 1 mm 3 . The in-gel Lys-C digestion was done as described (13). Each sample was dissolved in 5% acetonitrile, 2% formic acid and (5 l of 19 l) were used for LC-MS analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Differentially down-regulated phosphorylation of BAD at Ser-155 during HFD could be reversed by amorfrutin supplementation leading to phosphorylation levels similar as during LFD feeding. The light peak at m/z 706.78 is originated from the unlabeled mouse, whereas the heavy peak at m/z 715.79 represents the 13 C-lysine labeled reference mouse. Thus, ratios between LFD/HFD and HFDϩA1prev/HFD could be calculated.…”

Section: Fig 3 Protein Pathway Analysis Of Visceral White Adipose Tmentioning

confidence: 99%

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Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Meierhofer

Weidner

Hartmann

et al. 2013

Molecular & Cellular Proteomics

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Gaining understanding of common complex diseases and their treatments are the main drivers for life sciences. As we show here, comprehensive protein set analyses offer new opportunities to decipher functional molecular networks of diseases and assess the efficacy and side-effects of treatments in vivo. Using mass spectrometry, we quantitatively detected several thousands of proteins and observed significant changes in protein pathways that were (dys-) regulated in diet-induced obesity mice. Analysis of the expression and post-translational modifications of proteins in various peripheral metabolic target tissues including adipose, heart, and liver tissue generated functional insights in the regulation of cell and tissue homeostasis during high-fat diet feeding and medication with two antidiabetic compounds. Protein set analyses singled out pathways for functional characterization, and indicated, for example, early-on potential cardiovascular complication of the diabetes drug rosiglitazone. In vivo protein set detection can provide new avenues for monitoring complex disease processes, and for evaluating preclinical drug candidates. Molecular & Cellular Proteomics 12:

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Section: Methodsmentioning

confidence: 99%

Section: Fig 3 Protein Pathway Analysis Of Visceral White Adipose Tmentioning

confidence: 99%

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Meierhofer

Weidner

Hartmann

et al. 2013

Molecular & Cellular Proteomics

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“…12 After washing, the eluate consisted of the protein of interest bound to the interacting partners is then released with ethylene glycol tetraacetic acid (EGTA). 13 Copurifying proteins from the bound complex can be determined in two complementary ways. Each purified protein preparation is electrophoresed on an SDS polyacrylamide gel, stained with silver, and visible bands removed and identified by trypsin digestion and peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS.…”

Section: Mass Spectrometry Coupled Withmentioning

confidence: 99%

“…99 The basis of NMR spectroscopy is the property of many elements to have a nuclear magnetic moment. Stable isotopes of particular importance in biological macromolecules are 1 H, 13 C, or 15 N. When placed into a static magnetic field (B), the different nuclear spin states of these nuclei become quantized with energies proportional to their projection onto B. The energy difference depends on the type of nucleus, is proportional to field strength of the static magnet, and is dependent on the chemical environment of the nucleus.…”

Section: Nuclear Magnetic Resonance Spectroscopymentioning

confidence: 99%

How to Study Protein-protein Interactions

Podobnik¹,

Kraševec²,

Zavec³

et al. 2016

ACSi

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In memory of prof. dr. Janko Jamnik. AbstractPhysical and functional interactions between molecules in living systems are central to all biological processes. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Several powerful methodologies and techniques have been developed to study molecular interactions and thus help elucidate their nature and role in biology as well as potential ways how to interfere with them. All different techniques used in these studies have their strengths and weaknesses and since they are mostly employed in in vitro conditions, a single approach can hardly accurately reproduce interactions that happen under physiological conditions. However, complementary usage of as many as possible available techniques can lead to relatively realistic picture of the biological process. Here we describe several proteomic, biophysical and structural tools that help us understand the nature and mechanism of these interactions.

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“…Proteins were extracted and digested with trypsin according to Kaiser et al [13]. The peptide mixture was separated using a Zorbax 300SB-C18 (Agilent) column at a flow rate of 300 nl/min and analyzed on a QTRAP5500 (AB/Sciex) triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode.…”

Section: Immunoblotting and Antibodiesmentioning

confidence: 99%

The difference between rare and exceptionally rare: molecular characterization of ribose 5-phosphate isomerase deficiency

et al. 2010

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Ribose 5-phosphate isomerase (RPI) deficiency is an enzymopathy of the pentose phosphate pathway. It manifests with progressive leukoencephalopathy and peripheral neuropathy and belongs, with one sole diagnosed case, to the rarest human disorders. The single patient was found compound heterozygous for a RPI frameshift and a missense (RPI Ala61Val ) allele. Here, we report that two patient-derived cell lines differ in RPI enzyme activity, enzyme concentration, and mRNA expression. Furthermore, we present a transgenic yeast model, which exhibits metabolite-and enzyme-activity changes that correspond to the human syndrome and show that the decrease in RPI activity in patient cells is not fully attributable to the residue exchange. Taken together, our results demonstrate that RPI deficiency is caused by the combination of a RPI null allele with an allele that encodes for a partially active enzyme which has, in addition, cell-type-dependent expression deficits. We speculate that a low probability for comparable traits accounts for the rareness of RPI deficiency.

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Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

Cited by 35 publications

References 12 publications

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

How to Study Protein-protein Interactions

The difference between rare and exceptionally rare: molecular characterization of ribose 5-phosphate isomerase deficiency

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