Proteomics Core Facilities
FacilityThe Proteomics Core facility is a collaborative research enterprise that provides state-of-the-art proteomics services to researchers from South Dakota and the surrounding region.
Contact Us
Proteomics Core Facilities
Lee Medical Building
414 E. Clark St.
Vermillion, SD 57069
414 E. Clark St.
Vermillion, SD 57069
Office Hours
Monday - Friday
8:00 a.m. - 5:00 p.m.
8:00 a.m. - 5:00 p.m.
Our Facility
Supported by the National Institute of General Medical Sciences grant and a partnership between the Sanford School of Medicine and the South Dakota Biomedical Research Infrastructure Network (SD BRIN), the core facility provides researchers with the capability to rapidly analyze and identify protein expression patterns in their experimental systems.
Along with providing proteomic analysis, the core:
- Develops experimental design, protocols, data analysis and interpretation
- Provides consulting and advice in grant proposal, as well as data preparation to be submitted to proteomics journal according to the requirements
- Offers training in the use of common equipment such as the scanner, spot cutter, imaging software, technique and protocol issues and sample preparation
Equipment and Facility Use
Protein Identification
- This process identifies proteins from simple and complex mixtures from different samples’ matrices using discovery proteomics through bottom-up approach, peptide mass fingerprinting-MS or peptide fragment fingerprinting-MS/MS analysis.
- The samples come in gel or in solution digestion of simple and complex mixtures (shotgun proteomics). Typically, protein spots or bands excised from 1D or 2D gels or from in solution are digested with trypsin and/or trypsin/Lys-C and the peptides resolved using a 1D or 2D NanoAcquity ultra performance liquid chromatography.
- Tryptic peptides are desalted and concentrated using a reverse-phase trapping column and then resolved using a C18 reverse phase analytical column. The mass of peptides are determined using a nano-ESI Quadrupole-Time of Flight mass spectrometer in MS and MS/MS mode. The peptide masses are used to query various databases using several search engines, including ProteinLynx global server v3.0.3 Expression analysis, Mascot server and Proteome Discoverer to identify the most probable proteins in the sample.
We also provide:
- Protein characterization, including post-translational modifications and indirect protein-protein interaction analysis
- Protein quantification using label-free and labeled approaches
Peptide Mass Fingerprinting and Peptide Fragment Fingerprinting
- We train in peptide mass fingerprinting and peptide fragment fingerprinting through in-gel or in-solution digestion of simple and complex mixtures. Typically, protein spots or bands excised from 1D or 2D gels or from in-solution are digested with trypsin and/or Trypsin/Lys-C and the peptides resolved using a 1D or 2D NanoAcquity ultra performance liquid chromatography.
- Tryptic peptides are desalted and concentrated using a reverse-phase trapping column and then resolved using a C18 reverse phase analytical column. The mass of peptides are determined using a nano-ESI Quadrupole-Time of Flight mass spectrometer in MS and MS/MS mode. The peptide masses are used to query various databases, using ProteinLynx global server v3.0 Expression analysis or Mascot server to identify the most probable proteins in the sample.
Intact Protein Analysis
This type of analysis determines the molecular weight of an intact protein and can be used to identify post-translational modifications (phosphorylation, acetylation, etc.), proteolytic modifications and to determine the number of proteins in a sample. A typical procedure involves injecting the sample into the mass spectrometer using a regular electrospray or nano-electrospray configuration. Multiple charged spectra are generated and the spectra is deconvoluted using maximum entropy conversion software.
Equipment Training
We provide training in using the Typhoon 9410 scanner, spot cutter, image software analysis and basic excision tools for the cutter.
Consultation
We offer advice related to sample preparation, technique and protocol development or proteomics applications, as well as bioinformatics tools.
1D and 2D nanoAcquity Ultra Performance Liquid Chromatography
This is a liquid chromatography system that works in nano scale and allows you to separates small quantities of samples (fentomoles) using a nanoflow (200-400 nL/min) and also resolves complexes mixtures such as whole cell lysates, secretome and subcellular fractionation (nuclei and cytoplasm).
Ultimate RSLC3000 Ultra-High Performance Liquid Chromatography
This is a high-performance chromatography system with the capabilities to handle nano (50-1000 nL/min), capillary (1-10 ul/min) and micro flow settings (5-50 ul/min).
Synapt G1 HDMS
A hybrid Quadrupole-time of flight mass spectrometer with nano and regular spray ion sources and lockmass.
QExactive Plus
Orbitrap FTMS high resolution/mass accuracy (HR/MA) with enhanced resolution to 280,000 with a high-energy collision cell (HDC) with nano and regular spray ion source.
Typhoon 9410 Variable Mode Imager
A scanner with capabilities to work in fluorescence mode (four lasers and different filters), chemiluminescence and Phosphor imager (32P, 33P and 35S). Applications: gels, pvdf membranes, well plates immunohistochemistry, in situ hybridization and micro arrays.
ProteomeWork Spot Cutter
A robot picker arm with a digital camera that allows to perform the automatic gel spots or bands cut either through visible or UV lights.
MassPrep Workstation Multiprobe II
An automatic liquid handling system programmable to perform different protocols such as gel processing, protein digestion and peptides extraction.
Solid Phase Extraction
A system with manifold and a dedicated vacuum pump to clean up and concentrate samples from different biological matrices.
Zoom IEF Fractionator
Provides a method to reduce sample complexity, enrich low abundance proteins and increase the dynamic range of detection. Solution phase isoelectric focusing with the ZOOM IEF Fractionator provides reproducible separations. Fractionated samples are ready for further analysis by two dimensional gel electrophoresis, one dimensional gel electrophoresis or two dimensional liquid chromatography/mass spectrometry.
SPD1010 Integrated SpeedVac
A centrifuge to dry and concentrate aqueous or non-aggressive samples with speed and cost-efficiency.
Akta FPLC
Used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the mobile phase) and a porous solid (the stationary phase).
Expedeon GELFREE 8100 Fractionation System
An electrophoresis system for MW-based fractionation with liquid phase recovery. The instrument is comprised of single-use, eight-sample capacity cartridges and a benchtop GELFREE fractionation instrument. The sample (proteins) can be loaded into a chamber, concentrated into a tight band in a stacking gel and separated based in electrophoretic mobilities in a resolving gel. Subsequently, the proteins eluted from the column are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This kind of system provides a simple and fast fractionation, is easy to use, and allows recovering of the proteins in liquid phase without the necessity to extract them from the gel. The system can fractionate a wide range of MW from 3.5-500 kDa and provides at least four different options of percentage of polyacrylamide. The GELFREE 8100 is a useful tool to apply to the decreases of the complexity of the protein dynamic ranges present in complexes mixtures such as biological fluids, whole cell lysates and subcellular fractions.
1. Pizarro-Guajardo M, Ravanal MC, Paez MD, Callegari E, Paredes-Sabja D. Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291. Proteomics Clin Appl. 2018 Mar 23:e1700182. doi: 10.1002/prca.201700182. [Epub ahead of print] PubMed PMID: 29573213.
2: Dachineni R, Kumar DR, Callegari E, Kesharwani SS, Sankaranarayanan R, Seefeldt T, Tummala H, Bhat GJ. Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer. Int J Oncol. 2017 Dec;51(6):1661-1673. doi: 10.3892/ijo.2017.4167. Epub 2017 Oct 19. PubMed PMID: 29075787; PubMed Central PMCID: PMC5673027.
3: Oleas G, Callegari E, Sepúlveda R, Eyzaguirre J. Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp. Carbohydr Res. 2017 Apr 18;443-444:42-48. doi: 10.1016/j.carres.2017.03.014. Epub 2017 Mar 18. PubMed PMID: 28342968; PubMed Central PMCID: PMC5560272.
4: Oleas G, Callegari E, Sepulveda R, Eyzaguirre J. Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp. Insights Enzym Res. 2016;1(1). pii: 4. Epub 2016 Dec 12. PubMed PMID: 28828411; PubMed Central PMCID: PMC5562236.
5: Bonilla JO, Callegari EA, Delfini CD, Estevez MC, Villegas LB. Simultaneous chromate and sulfate removal by Streptomyces sp. MC1. Changes in intracellular protein profile induced by Cr(VI). J Basic Microbiol. 2016 Nov;56(11):1212-1221. doi: 10.1002/jobm.201600170. Epub 2016 Jun 10. PubMed PMID: 27283205.
6: Zhang L, Hapon MB, Goyeneche AA, Srinivasan R, Gamarra-Luques CD, Callegari EA, Drappeau DD, Terpstra EJ, Pan B, Knapp JR, Chien J, Wang X, Eyster KM, Telleria CM. Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors. Mol Oncol. 2016 Aug;10(7):1099-117. doi: 10.1016/j.molonc.2016.05.001. Epub 2016 May 17. PubMed PMID: 27233943; PubMed Central PMCID: PMC5240778.
7: Callegari EA, Chaussee MS. The Streptococcal Proteome. 2016 Feb 10. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016-. Available from http://www.ncbi.nlm.nih.gov/books/NBK333416/ PubMed PMID: 26866219.
8: Mardones W, Callegari E, Eyzaguirre J. Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization. Fungal Biol. 2015 Dec;119(12):1267-1278. doi: 10.1016/j.funbio.2015.09.009. Epub 2015 Oct 14. PubMed PMID: 26615749.
9: Callegari EA. Shotgun Proteomics Analysis of Estrogen Effects in the Uterus Using Two-Dimensional Liquid Chromatography and Tandem Mass Spectrometry. Methods Mol Biol. 2016;1366:131-148. doi: 10.1007/978-1-4939-3127-9_11. PubMed PMID: 26585132; PubMed Central PMCID: PMC5364499.
10: Dong G, Gross K, Qiao F, Ferguson J, Callegari EA, Rezvani K, Zhang D, Gloeckner CJ, Ueffing M, Wang H. Calretinin interacts with huntingtin and reduces mutant huntingtin-caused cytotoxicity. J Neurochem. 2012 Nov;123(3):437-46. doi: 10.1111/j.1471-4159.2012.07919.x. Epub 2012 Sep 10. PubMed PMID: 22891683.
11: McDowell EJ, Callegari EA, Malke H, Chaussee MS. CodY-mediated regulation of Streptococcus pyogenes exoproteins. BMC Microbiol. 2012 Jun 21;12:114. doi: 10.1186/1471-2180-12-114. PubMed PMID: 22721528; PubMed Central PMCID: PMC3438106.
12: Dong G, Callegari E, Gloeckner CJ, Ueffing M, Wang H. Mass spectrometric identification of novel posttranslational modification sites in Huntingtin. Proteomics. 2012 Jun;12(12):2060-4. doi: 10.1002/pmic.201100380. PubMed PMID: 22623107; PubMed Central PMCID: PMC3845347.
13: Eduardo Callegari and Mario Navarrete. The Use of Mass Spectrometry for Characterization of Fungal Secretomes Part of the book: Tandem Mass Spectrometry, applications and principles.Editor: Jeevan Praisan, Intech, chapter 10, 221-234, February 29th 2012 ISBN 978-953-51-0141-3 DOI: 10.5772/30908.
14: Sadhu SS, Callegari E, Zhao Y, Guan X, Seefeldt T. Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1. J Enzyme Inhib Med Chem. 2013 Jun;28(3):456-62. doi: 10.3109/14756366.2011.649267. Epub 2012 Feb 3. PubMed PMID: 22299579; PubMed Central PMCID: PMC4310727.
15: Navarrete M, Callegari E, Eyzaguirre J. The effect of acetylated xylan and sugar beet pulp on the expression and secretion of enzymes by Penicillium purpurogenum. Appl Microbiol Biotechnol. 2012 Jan;93(2):723-41. doi: 10.1007/s00253-011-3744-y. Epub 2011 Dec 11. PubMed PMID: 22159660.
16: Dong G, Callegari EA, Gloeckner CJ, Ueffing M, Wang H. Prothymosin-α interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture. J Biol Chem. 2012 Jan 6;287(2):1279-89. doi: 10.1074/jbc.M111.294280. Epub 2011 Nov 22. PubMed PMID: 22110140; PubMed Central PMCID: PMC3256907.
17: Gonzalez-Vogel A, Eyzaguirre J, Oleas G, Callegari E, Navarrete M. Proteomic analysis in non-denaturing condition of the secretome reveals the presence of multienzyme complexes in Penicillium purpurogenum. Appl Microbiol Biotechnol. 2011 Jan;89(1):145-55. doi: 10.1007/s00253-010-2953-0. Epub 2010 Oct 23. PubMed PMID: 20972675.
18: Ravanal MC, Callegari E, Eyzaguirre J. Novel bifunctional alpha-L-arabinofuranosidase/xylobiohydrolase (ABF3) from Penicillium purpurogenum. Appl Environ Microbiol. 2010 Aug;76(15):5247-53. doi: 10.1128/AEM.00214-10. Epub 2010 Jun 18. PubMed PMID: 20562284; PubMed Central PMCID: PMC2916492.
19: Gao X, Wan F, Mateo K, Callegari E, Wang D, Deng W, Puente J, Li F, Chaussee MS, Finlay BB, Lenardo MJ, Hardwidge PR. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function. PLoS Pathog. 2009 Dec;5(12):e1000708. doi: 10.1371/journal.ppat.1000708. Epub 2009 Dec 24. PubMed PMID: 20041225; PubMed Central PMCID: PMC2791202.
20: Mo B, Callegari E, Telefont M, Renner KJ. Estrogen regulation of proteins in the rat ventromedial nucleus of the hypothalamus. J Proteome Res. 2008 Nov;7(11):5040-8. doi: 10.1021/pr8005974. Epub 2008 Oct 8. PubMed PMID: 18841879; PubMed Central PMCID: PMC2633946.
21: Fernández D, Bertoldi MV, Gómez L, Morales A, Callegari E, Lopez LA. Identification and characterization of Myosin from rat testicular peritubular myoid cells. Biol Reprod. 2008 Dec;79(6):1210-8. doi: 10.1095/biolreprod.107.066472. Epub 2008 Aug 20. PubMed PMID: 18716291; PubMed Central PMCID: PMC2586039.
22: Fanelli MA, Montt-Guevara M, Diblasi AM, Gago FE, Tello O, Cuello-Carrión FD, Callegari E, Bausero MA, Ciocca DR. P-cadherin and beta-catenin are useful prognostic markers in breast cancer patients; beta-catenin interacts with heat shock protein Hsp27. Cell Stress Chaperones. 2008 Summer;13(2):207-20. doi: 10.1007/s12192-007-0007-z. Epub 2008 Mar 5. PubMed PMID: 18320359; PubMed Central PMCID: PMC2673888.
23: Ristic Z, Momcilović I, Fu J, Callegari E, DeRidder BP. Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase. J Plant Physiol. 2007 Dec;164(12):1564-71. Epub 2007 Sep 4. PubMed PMID: 17766005.
24: Chaussee MA, Dmitriev AV, Callegari EA, Chaussee MS. Growth phase-associated changes in the transcriptome and proteome of Streptococcus pyogenes. Arch Microbiol. 2008 Jan;189(1):27-41. Epub 2007 Jul 31. PubMed PMID: 17665172.
25: Mo B, Callegari E, Telefont M, Renner KJ. Proteomic analysis of the ventromedial nucleus of the hypothalamus (pars lateralis) in the female rat. Proteomics. 2006 Nov;6(22):6066-74. PubMed PMID: 17051637.
26: Chaussee MA, McDowell EJ, Rieck LD, Callegari EA, Chaussee MS. Proteomic analysis of a penicillin-tolerant rgg mutant strain of Streptococcus pyogenes. J Antimicrob Chemother. 2006 Oct;58(4):752-9. Epub 2006 Aug 5. PubMed PMID: 16891633.
27: Chaussee MA, Callegari EA, Chaussee MS. Rgg regulates growth phase-dependent expression of proteins associated with secondary metabolism and stress in Streptococcus pyogenes. J Bacteriol. 2004 Nov;186(21):7091-9. PubMed PMID: 15489420; PubMed Central PMCID: PMC523193.
28: Rao D, Momcilovic I, Kobayashi S, Callegari E, Ristic Z. Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu. Eur J Biochem. 2004 Sep;271(18):3684-92. PubMed PMID: 15355346.
For further information on Proteomics Core capabilities and use, please contact Eduardo Callegari, Proteomics Core Facility Manager, at (605)-677-5530 or Eduardo.Callegari@usd.edu. You can also reach out to Daniela Paez, Researcher Associate II, at (605)-658-6463 or Daniela.Paez@usd.edu.
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