捆绑SM社区

捆绑SM社区.CA / Jewish General Hospital Proteomics Centre

Services

We offer a range of services such as sample preparation, mass spectrometry measurement as well as data analysis and presentation. Our methods can be applied to a wide variety of samples including purified proteins and protein complexes, cells, tissues and biofluids.

1) Identification of proteins and their post-translational modifications (PTMs)
We offer mass spectrometry-based identification of proteins and PTMs within a given sample, including acetylation, glycosylation, phosphorylation, proteolytic cleavage, and ubiquitination. Only high confidence results are reported using state-of-the-art data analysis tools and strategies.(1-6)
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2) Relative quantitative proteomics and phosphoproteomics for discovery
In-depth analysis of cells, tissues and biofluids will lead to the identification of regulated proteins and pathways. Depending on sample type, up to 10,000 proteins and more than 10,000 phosphorylation sites can be quantified from as little as 25 碌g (proteome) to 100 碌g (phosphoproteome) of total protein per lysate. Only high confidence identifications using state-of-the-art data analysis tools and strategies are reported. Significantly regulated proteins and phosphorylation sites will be reported using fold-change cutoffs based on the data distribution and statistical tests. Visualization of regulated pathways etc. is available on request. (7-9)
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3) Targeted quantitative mass spectrometry
We have developed a large number of assays for precise absolute quantification of selected proteins using stable isotope-labeled reference standard(SIS) peptides in combination with targeted mass spectrometry (Multiple Reaction Monitoring, MRM; Parallel Reaction Monitoring, PRM).
These highly-specific assays allow the precise and sensitive determination of protein concentrations in mole per volume/cell/weight, down to the attomolar range. MRM and PRM can be used for high-throughput (multiplexed) quantification of proteins without the need for antibodies. Moreover, they are more precise and specific than Western blots. Targeted MS using SIS peptides allows the determination of phosphorylation stoichiometries. (10-13)

4) Multi-OMICS
Our MultiOMICS service consists of the combined analysis of the proteome, metabolome and lipidome from a single sample (no replicates, all from one vial) using our SIMPLEX (Simultaneous Metabolite, Protein, Lipid Extraction) strategy. The complementary datasets provide a view of the interconnectivity of different biomolecular classes, which provide an extremely detailed view of the phenotype of the sample. (14)


5) Protein-protein interaction studies / structural proteomics
We employ chemical cross-linking of proteins to identify interaction partners and protein complex components using mass spectrometry. This approach also allows for prediction of protein structures not easily determined by X-ray crystallography or other 鈥渢raditional鈥� approaches. (15-18)


6) Immuno-MS assays
Our immuno-MS assays are based on immuno-enrichment of targets using anti-peptide and anti-protein antibodies, combined with mass spectrometry for the highly sensitive and quantification of proteins and their PTMs, from clinically relevant sample types (e.g., needle biopsies). Anti-peptide immuno-MS assays are established for AKT1, AKT2, and under development for P13K, PD-L1 and PTEN (peptide+protein). (19-22)


7) Metabolomics
As a member of the Metabolomics Innovation Centre (TMIC), we offer quantitative MS-based metabolomics assays for hundreds of small molecules, with a variety of panels representing different metabolic pathways or classes of molecules.

List of Assays:

13-Fluxome Analysis

Antibiotics Assay

Antivirals Assay

CYP Cocktail

Drug MetID

HCQ/Az Assay

Plasma Free Metanephrines Assay

Uracil Assay

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8)听Plasma free metanephrines

Quantitation of plasma free metanephrines and 3-methoxytyramine which can be used to help diagnose catecholamine secreting tumours (pheochromocytomas/paragangliomas and neuroblastomas).

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DEVELOPED ASSAYS

1. Name of Assay: Akt1 / Akt2 / phospho-Akt1 s473 / phospho-Akt2 s474

Description of Assay: iMALDI quantitation of Akt isoforms using proteotypic peptides. This assay is validated for use in a variety of sample types including cell lysates, fresh-frozen tissue, FFPE tissue. Phosphorylation stoichiometry for selected peptides is determined via measurement of the target peptide with and without dephosphorylation. Compatible with samples with limited material and compatible with high throughput for large studies.
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2. Name of Assay: Cytochrome P450 enzyme activity assay for CYP1A2 and CYP3A4 through measurement of caffeine and midazolam and their metabolites

Description of Assay: This metabolomics assay enables assessment of the CYP1A2 and CYP3A4 enzyme activity in clinical subjects via plasma sample or dried blood spot, collected following administration of a representative drug. Please see Bosilkovska M, Samer CF, Deglon J, Rebsamen M, Staub C, Dayer P, Walder B, Desmeules JA, Daali Y. Geneva cocktail for cytochrome p450 and P鈥恎lycoprotein activity assessment using dried blood spots. ClInICAl PhArMACologY & TherAPeuTICS. 2014 Sep;96(3):349-59 for further details. The assay may be readily extended to other CYPs through the addition of targeted assays for other exogenous compounds, as previously published.
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3.听Name of Assay: Comprehensive proteome analysis from formalin fixed specimens

Description of Assay: MS-based proteomic analysis of FFPE material has increasingly gained attention and allows deep proteome profiling from small amounts of protein starting material. Quantitative proteomics enables the identification of differentially expressed proteins and aberrant signaling pathway activity. This is most-relevant phenotypic information that can be extracted neither from conventional genomic screening nor from IHC staining and that can be in discordance with genomic information. Thus, proteomics of FFPE tissues enables the retrospective and 鈥榰nbiased鈥� discovery of (previously unknown) protein biomarkers and enables the identification of molecular mechanisms of disease from rare clinical specimens that were collected over a long period of time.
Our streamlined non-hazardous FFPE-proteomics workflow for use in basic and clinical research allows the quantification and identification of more than 1500 proteins (identified with at least 2 protein unique peptides and FDR of 1%) from a single FFPE core of 1-mm diameter and less than 1 mg dry-weight before deparaffinization. Clinically relevant data from very low starting material (>0.5 碌g total protein) can be available within 6 hours, including sample processing and instrument time.
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4. Name of Assay: Quantitation of cancer signaling proteins

Description of Assay: The concentrations of low abundance cancer signaling proteins are determined using targeted mass spectrometry and stable isotope labeled standard peptides. The assays include the following proteins

  • mTOR
  • PTEN
  • PI3K
  • AKT1
  • AKT2

which can be quantified in a multiplexed fashion. The assays can be applied to human biofluids, cells, fresh frozen and FFPE tissues, requiring only low sample input. The high precision (CVs<15%) and standardization of the technology are ideal for large-scale studies and inter-laboratory comparisons.

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5.听Name of Assay: Quantitation of PD-L1 axis proteins

Description of Assay:The concentrations of proteins involved in PD-L1 signaling are determined using targeted mass spectrometry and stable isotope labeled standard peptides. The assays include the following proteins

  • PD-L1
  • PD-L2
  • PD-1
  • LCK
  • ZAP70
  • NT5E (CD73)

which can be quantified in a multiplexed fashion. The assays can be applied to human biofluids, cells, fresh frozen and FFPE tissues, requiring only low sample input. The high precision (CVs<15%) and standardization of the technology are ideal for large-scale studies and inter-laboratory comparisons.

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6.听Name of Assay: Quantitation of therapeutic monoclonal antibodies / therapeutic drug monitoring

Description of Assay:The concentrations of therapeutic monoclonal antibodies (mAbs) in plasma are determined using targeted mass spectrometry and stable isotope labeled standard peptides. The assays include the following antibodies

  • Adalimumab
  • Bevacizumab
  • Infliximab
  • Rituximab
  • Trastuzumab

which can be quantified in a multiplexed fashion. The assays require only 10 碌L of human plasma. The high precision (CVs<15%) and standardization of the technology are ideal for large-scale studies and inter-laboratory comparisons.

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7.听Name of Assay: Quantitation of 270+ plasma proteins

Description of Assay: The concentrations of >270 proteins in human plasma are determined using targeted mass spectrometry and stable isotope labeled standard peptides. The assays include >70 FDA-approved biomarkers. The high precision (CVs<15%) and standardization of the technology are ideal for large-scale studies and inter-laboratory comparisons.

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References听

1. Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability. V枚gtle, F.-N., Wortelkamp, S., Zahedi, R.P., Becker, D., Leidhold, C., Gevaert, K., Kellermann, J., Voos, W., Sickmann, A., Pfanner, N., Meisinger, C., 2009. Cell 139, 428鈥�439.


2. Simple, scalable, and ultrasensitive tip-based identification of protease substrates. Shema, G., Nguyen, M.T.N., Solari, F.A., Loroch, S., Venne, A.S., Kollipara, L., Sickmann, A., Verhelst, S.H.L., Zahedi, R.P., 2018. Mol. Cell. Proteomics MCP 17, 826鈥�834.


3. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Saita, S., Nolte, H., Fiedler, K.U., Kashkar, H., Venne, A.S., Zahedi, R.P., Kr眉ger, M., Langer, T., 2017. Nat. Cell Biol. 19, 318鈥�328.


4. Effective Assignment of 伪2,3/伪2,6-Sialic Acid Isomers by LC-MS/MS-Based Glycoproteomics. Pett, C., Nasir, W., Sihlbom, C., Olsson, B.-M., Caixeta, V., Schorlemer, M., Zahedi, R.P., Larson, G., Nilsson, J., Westerlind, U., 2018. Angew. Chem. Int. Ed Engl. 57, 9320鈥�9324.


5. Enhanced N-glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes. Lewandrowski, U., Zahedi, R.P., Moebius, J., Walter, U., Sickmann, A., 2007. Mol. Cell. Proteomics MCP 6, 1933鈥�1941.


6. Regulation of mitochondrial protein import by cytosolic kinases. Schmidt, O., Harbauer, A.B., Rao, S., Eyrich, B., Zahedi, R.P., Stojanovski, D., Sch枚nfisch, B., Guiard, B., Sickmann, A., Pfanner, N., Meisinger, C., 2011. Cell 144, 227鈥�239.

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7. Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin. Gonczarowska-Jorge, H., Loroch, S., Dell鈥橝ica, M., Sickmann, A., Roos, A., Zahedi, R.P., 2017. Anal. Chem. 89, 13137鈥�13145.


8. Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways. Beck, F., Geiger, J., Gambaryan, S., Veit, J., Vaudel, M., Nollau, P., Kohlbacher, O., Martens, L., Walter, U., Sickmann, A., Zahedi, R.P., 2014. Blood 123, e1鈥揺10.


9. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Beck, F., Geiger, J., Gambaryan, S., Solari, F.A., Dell鈥橝ica, M., Loroch, S., Mattheij, N.J., Mindukshev, I., P枚tz, O., Jurk, K., Burkhart, J.M., Fufezan, C., Heemskerk, J.W.M., Walter, U., Zahedi, R.P., Sickmann, A., 2017. Blood 129, e1鈥揺12.

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10. The mTOR and PP2A Pathways Regulate PHD2 Phosphorylation to Fine-Tune HIF1伪 Levels and Colorectal Cancer Cell Survival under Hypoxia. Di Conza, G., Trusso Cafarello, S., Loroch, S., Mennerich, D., Deschoemaeker, S., Di Matteo, M., Ehling, M., Gevaert, K., Prenen, H., Zahedi, R.P., Sickmann, A., Kietzmann, T., Moretti, F., Mazzone, M., 2017. Cell Rep. 18, 1699鈥�1712.

11. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma. Kuzyk, M.A., Smith, D., Yang, J., Cross, T.J., Jackson, A.M., Hardie, D.B., Anderson, N.L., Borchers, C.H., 2009. Mol. Cell. Proteomics MCP 8, 1860鈥�1877.


12. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Addona, T.A., Abbatiello, S.E., Schilling, B., Skates, S.J., Mani, D.R., Bunk, D.M., Spiegelman, C.H., Zimmerman, L.J., Ham, A.-J.L., Keshishian, H., Hall, S.C., Allen, S., Blackman, R.K., Borchers, C.H., Buck, C., Cardasis, H.L., Cusack, M.P., Dodder, N.G., Gibson, B.W., Held, J.M., Hiltke, T., Jackson, A., Johansen, E.B., Kinsinger, C.R., Li, J., Mesri, M., Neubert, T.A., Niles, R.K., Pulsipher, T.C., Ransohoff, D., Rodriguez, H., Rudnick, P.A., Smith, D., Tabb, D.L., Tegeler, T.J., Variyath, A.M., Vega-Montoto, L.J., Wahlander, A., Waldemarson, S., Wang, M., Whiteaker, J.R., Zhao, L., Anderson, N.L., Fisher, S.J., Liebler, D.C., Paulovich, A.G., Regnier, F.E., Tempst, P., Carr, S.A., 2009. Nat. Biotechnol. 27, 633鈥�641.


13. MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Domanski, D., Percy, A.J., Yang, J., Chambers, A.G., Hill, J.S., Freue, G.V.C., Borchers, C.H., 2012. Proteomics 12, 1222鈥�1243.

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14. Simultaneous Metabolite, Protein, Lipid Extraction (SIMPLEX): A Combinatorial Multimolecular Omics Approach for Systems Biology. Coman, C., Solari, F.A., Hentschel, A., Sickmann, A., Zahedi, R.P., Ahrends, R., 2016.听Mol. Cell. Proteomics听MCP 15, 1453鈥�1466.

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15. Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes. Petrotchenko, E.V., Olkhovik, V.K., Borchers, C.H., 2005. Mol. Cell. Proteomics MCP 4, 1167鈥�1179.


16. Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism. Solomonson, M., Setiaputra, D., Makepeace, K.A.T., Lameignere, E., Petrotchenko, E.V., Conrady, D.G., Bergeron, J.R., Vuckovic, M., DiMaio, F., Borchers, C.H., Yip, C.K., Strynadka, N.C.J., 2015. Struct. Lond. Engl. 1993 23, 571鈥�583. .


17. Super Spy variants implicate flexibility in chaperone action. Quan, S., Wang, L., Petrotchenko, E.V., Makepeace, K.A., Horowitz, S., Yang, J., Zhang, Y., Borchers, C.H., Bardwell, J.C., 2014. eLife 3, e01584.


18. Architecture of the RNA polymerase II-Mediator core initiation complex. Plaschka, C., Larivi猫re, L., Wenzeck, L., Seizl, M., Hemann, M., Tegunov, D., Petrotchenko, E.V., Borchers, C.H., Baumeister, W., Herzog, F., Villa, E., Cramer, P., 2015. Nature 518, 376鈥�380.

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19. An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis. Jiang, J., Parker, C.E., Fuller, J.R., Kawula, T.H., Borchers, C.H., 2007. Anal. Chim. Acta 605, 70鈥�79.


20. Towards the development of an immuno MALDI (iMALDI) mass spectrometry assay for the diagnosis of hypertension. Reid, J.D., Holmes, D.T., Mason, D.R., Shah, B., Borchers, C.H., 2010. J. Am. Soc. Mass Spectrom. 21, 1680鈥�1686.


21. Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension. Camenzind, A.G., van der Gugten, J.G., Popp, R., Holmes, D.T., Borchers, C.H., 2013. Clin. Proteomics 10, 20.


22. Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors. Popp, R., Li, H., LeBlanc, A., Mohammed, Y., Aguilar-Mahecha, A., Chambers, A.G., Lan, C., Poetz, O., Basik, M., Batist, G., Borchers, C.H., 2017. Anal. Chem. 89, 10592鈥�10600.

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