Development of an LC-MALDI method for the analysis of protein complexes

Zhen, Yuejun; Xu, Nafei; Richardson, Bonnie; Becklin, Robert R.; Savage, Justin R.; Blake, Kelly D.; Peltier, John M.

doi:10.1016/j.jasms.2004.02.004

Cited by 75 publications

(45 citation statements)

References 56 publications

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“…Erastin A6 and B2 were linked to a solid-phase resin, which was then incubated with BJ-TERT/LT/ST/RAS V12 or BJ-TERT cell lysate and washed before protein was eluted and then digested as described 23 . Reverse-phase high-performance liquid chromatography was performed before samples were analysed using both a 4700 Proteomics Analyser matrixassisted laser desorption/ionization-time of flight (TOF)/TOF (TOF/TOF; Applied Biosystems) and a Q Trap (AB/MDS Sciex).…”

Section: Methods Summary Vdac Identification Using Pull-down Assaymentioning

confidence: 99%

RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

Yagoda

Rechenberg²,

Zaganjor

et al. 2007

Nature

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Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs 1 . Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)-a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions N.Y. designed and performed the RNAi and VDAC overexpression, quantitative PCR, erastin analogue viability and chemical characterization experiments. E.Z. performed two-dimensional western analysis, PARP-1 and pro-caspase-3 cleavage, and cytochrome c release experiments. E.Z. and N.Y. performed transmission electron microscopy experiments. A.J.B., D.J.F. and N.Y. performed the NADH oxidation and direct binding experiments. W.S.Y. characterized sensitivity to erastin in the BJderived cell series. A.J.W. performed the MEK1/2 inhibitor experiment. I.S. and A.J.B. synthesized erastin analogues. R.S. and S.L.L. provided BRAF shRNAs, analysis of BRAF knockdown and the phospho-ERK western analysis. J.M.P., J.J.B. and S.S. were responsible for setting up the technology platform to pull down proteins binding to small molecule compounds. M.v.R. and J.M.P. performed the pull-down experiments. J.J.B., J.M.P. and S.S. designed, reviewed and supervised the pull-down experiments, and contributed to the analysis of the data. B.R.S. conceived of and supervised the project, designed and analysed experiments, and performed the anti-oxidant studies. B.R.S. and N.Y. prepared the manuscript. (Fig. 1a , Supplementary Fig. 1 and ref. 3 ). This cell death was not dependent on the rate of cell division, nor was it idiosyncratic to these cells ( Fig. 1a and Supplementary Fig. 2), because cell lines engineered in a similar way responded similarly. Author InformationWe found th...

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Section: Methods Summary Vdac Identification Using Pull-down Assaymentioning

confidence: 99%

RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

Yagoda

Rechenberg²,

Zaganjor

et al. 2007

Nature

Self Cite

1,303

1,133

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“…Although post source decay (PSD) analysis [19] represents a pioneering way of simulating a tandem analyzer, the difficulty of reconstructing fragmentation spectra has precluded its being used for extensive peptide sequencing studies. The advent of true tandem TOF instruments [20,21] has increased the interest in MALDI as an ionization method and has led to the development of new MALDI-based proteomics workflows, notably LC-MALDI [22]. One of these new analyzers, the MALDI TOF/TOF [20] has unique fragmentation patterns where the tandem spectra are a combination of the products of both high-energy CID and lower-energy PSD.…”

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confidence: 99%

Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF

Campbell¹,

Vestal²,

Blank

et al. 2007

J. Am. Soc. Mass Spectrom.

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The effects of laser fluence on ion formation in MALDI were studied using a tandem TOF mass spectrometer with a Nd-YAG laser and ␣-cyano hydrocinnamic acid matrix. Leucine enkephalin ionization and fragmentation were followed as a function of laser fluence ranging from the threshold of ion formation to the maximum available, that is, about 280 -930 mJ/mm 2 . The most notable finding was the appearance of immonium ions at fluence values close to threshold, increasing rapidly and then tapering in intensity with the appearance of typical backbone fragment ions. The data suggest the presence of two distinct environments for ion formation. One is associated with molecular desorption at low values of laser fluence that leads to extensive immonium ion formation. The second becomes dominant at higher fluences, is associated initially with backbone type fragments, but, at the highest values of fluence, progresses to immonium fragments. This second environment is suggestive of ion desorption from large pieces of material ablated from the surface. Arrhenius rate law considerations were used to estimate temperatures associated with the onset of these two processes. (J Am Soc Mass Spectrom 2007, 18, 607-616)

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“…This method can be extended by coupling liquid chromatography with matrix-assisted laser-induced ionizationtandem time of flight (MALDI-TOF/TOF) MS. Relative and absolute quantitation of proteins by MS is possible with either labeled (Ross et al 2004;Zhen et al 2004) or label-free (Ono et al 2006) technologies. 1 The maturation of both microarray and proteomic technologies has led to a small number of wholetranscriptome-proteome comparisons (Gygi et al 1999;Valenzuela et al 2003;McRedmond et al 2004;Ribeiro et al 2004;Ruse et al 2004;Mijalski et al 2005;Pastorelli et al 2006).…”

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confidence: 99%

Applying Gene Expression, Proteomics and Single-Nucleotide Polymorphism Analysis for Complex Trait Gene Identification

et al. 2008

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Previous quantitative trait locus (QTL) analysis of an intercross involving the inbred mouse strains NZB/ BlNJ and SM/J revealed QTL for a variety of complex traits. Many QTL have large intervals containing hundreds of genes, and methods are needed to rapidly sort through these genes for probable candidates. We chose nine QTL: the three most significant for high-density lipoprotein (HDL) cholesterol, gallstone formation, and obesity. We searched for candidate genes using three different approaches: mRNA microarray gene expression technology to assess .45,000 transcripts, publicly available SNPs to locate genes that are not identical by descent and that contain nonsynonymous coding differences, and a massspectrometry-based proteomics technology to interrogate nearly 1000 proteins for differential expression in the liver of the two parental inbred strains. This systematic approach reduced the number of candidate genes within each QTL from hundreds to a manageable list. Each of the three approaches selected candidates that the other two approaches missed. For example, candidate genes such as Apoa2 and Acads had differential protein levels although the mRNA levels were similar. We conclude that all three approaches are important and that focusing on a single approach such as mRNA expression may fail to identify a QTL gene.

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Development of an LC-MALDI method for the analysis of protein complexes

Cited by 75 publications

References 56 publications

RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF

Applying Gene Expression, Proteomics and Single-Nucleotide Polymorphism Analysis for Complex Trait Gene Identification

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