Mayo Proteomics Research Center

Rochester, MN, United States

Mayo Proteomics Research Center

Rochester, MN, United States
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Eckel-Passow J.E.,Rochester College | Mahoney D.W.,Rochester College | Oberg A.L.,Rochester College | Zenka R.M.,Mayo Proteomics Research Center | And 3 more authors.
Journal of Proteomics and Bioinformatics | Year: 2010

Motivation: Interpreting and quantifying labeled mass-spectrometry data is complex and requires automated algorithms, particularly for large scale proteomic profiling. Here, we propose the use of bi-linear regression to quantify relative abundance across the elution profile in a unified model. The bi-linear regression model takes advantage of the fact that while peptides differ in overall abundance across the elution profile multiplicatively, the relative abundance between the mixed samples remains constant across the elution profile. We describe how to apply bi-linear regression models to 18O stable-isotope labeled data, which allows for the direct comparison of two samples simultaneously. Interpretation of model parameters is also discussed. The incorporation rate of the labeling isotope is estimated as part of the modeling process and can be used as a measure of data quality. Application is demonstrated in a controlled experiment as well as in a complex mixture. Results: Bi-linear regression models allow for more precise and accurate estimates of abundance, in comparison to methods that treat each spectrum independently, by taking into account the abundance of the molecule throughout the entire elution profile, with precision increased by one-to-two orders of magnitude.


Corena-McLeod M.,Neuropsycopharmacology Laboratory | Walss-Bass C.,University of Texas Health Science Center at San Antonio | Oliveros A.,Neuropsycopharmacology Laboratory | Gordillo Villegas A.,University of Florida | And 7 more authors.
PLoS ONE | Year: 2013

Background:Mitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level.Hypothesis:Mitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement.Methods:Synaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry.Results:Lithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation.Conclusions:Mood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs. © 2013 Corena-McLeod et al.


Mahoney D.W.,Mayo Medical School | Therneau T.M.,Mayo Medical School | Heppelmann C.J.,Mayo Proteomics Research Center | Higgins L.,University of Minnesota | And 6 more authors.
Journal of Proteome Research | Year: 2011

Shotgun proteomics via mass spectrometry (MS) is a powerful technology for biomarker discovery that has the potential to lead to noninvasive disease screening mechanisms. Successful application of MS-based proteomics technologies for biomarker discovery requires accurate expectations of bias, reproducibility, variance, and the true detectable differences in platforms chosen for analyses. Characterization of the variability inherent in MS assays is vital and should affect interpretation of measurements of observed differences in biological samples. Here we describe observed biases, variance structure, and the ability to detect known differences in spike-in data sets for which true relative abundance among defined samples were known and were subsequently measured with the iTRAQ technology on two MS platforms. Global biases were observed within these data sets. Measured variability was a function of mean abundance. Fold changes were biased toward the null and variance of a fold change was a function of proteinmass and abundance. The information presented herein will be valuable for experimental design and analysis of the resulting data. © 2011 American Chemical Society.


Chen Y.,Molecular Therapeutics | Snyder M.R.,Mayo Medical School | Zhu Y.,Mayo Proteomics Research Center | Tostrud L.J.,Mayo Medical School | And 3 more authors.
Clinical Chemistry | Year: 2011

BACKGROUND: α-1-Antitrypsin (A1AT) deficiency results from a genetic disorder at 2 common loci. Diagnosis requires quantification of A1AT and subsequent identification of the specific variant. The current algorithm of laboratory testing for the diagnosis of A1AT deficiency uses a combination of quantification (nephelometry), genotyping, and/or phenotyping. We developed a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of A1AT and identification of the 2 most common deficiency alleles present in 95% of the patients with A1AT deficiency. METHOD: Serum samples (n = 40) were digested with trypsin, and appropriate 13C/ 15N-labeled standard peptides were added. We performed LC-MS/MS analysis with a 0.5- by 150-mm C18 column and H 2O:acetonitrile: n-propanol:formic acid (A:98:1:1:0.2 and B:10:80:10:0.2; flow 12 μL/min) mobile phase in positive ion mode on a TSQ Quantum triple quadrupole MS system. We measured the A1AT concentration by comparison to a calibration curve and determined the phenotype by the presence or absence of variant peptides. We compared the results to the current phenotyping assay by isoelectric focusing (IEF) and the immunonephelometry quantitative assay. RESULTS: For A1AT allele detection, in 39 of 40 samples the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis. The single discrepant result was rerun by IEF at a lower dilution, and the results were in concordance. The A1AT quantification by LC-MS/MS also compared favorably with nephelometry. CONCLUSIONS: The LC-MS/MS method correlates well with current phenotyping and nephelometric assays and has the potential to improve the laboratory diagnosis of genetic A1AT deficiency. © 2011 American Association for Clinical Chemistry.


Salameh M.A.,Mayo Clinic Cancer Center | Robinson J.L.,Mayo Clinic Cancer Center | Navaneetham D.,Temple University | Sinha D.,Temple University | And 3 more authors.
Journal of Biological Chemistry | Year: 2010

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg15-Ala16 reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Devarajan-Ketha H.,Mayo Medical School | Craig T.A.,Mayo Medical School | Madden B.J.,Mayo Proteomics Research Center | Robert Bergen H.,Mayo Medical School | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2012

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1. M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone. © 2011 Elsevier Inc.


Pike G.M.,Mayo Medical School | Madden B.J.,Mayo Proteomics Research Center | Melder D.C.,Mayo Medical School | Charlesworth M.C.,Mayo Proteomics Research Center | Federspiel M.J.,Mayo Medical School
Journal of Biological Chemistry | Year: 2011

Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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