Mass Spectrometry and Tandem MS/MS
Instrumentation and Services Available
An ABSciexTripleTOF 5600+, an ABSciex 5800 Proteomics Analyzer (MALDI TOF-TOF), a Waters Synapt HDMS with Ion Mobility capacities, an MDS-Sciex 4000 QTrap, and a Voyager DE-PRO (Perseptive Biosystems) are available for analyses of protein, peptide, lipid, small molecule, and oligonucleotide samples. Analysis of digests of highly complex mixtures, such as whole cell lysates or serum proteins, commonly yield identification of 600-6000 proteins from single samples, & identification of proteins purified by column chromatography, by metal affinity chromatography, by gel electrophoresis, or from PVDF/nitrocellulose membranes are also available, e.g., detection of phosphoproteins and other post-translational modifications, isozyme analysis, & identification of disulfide bonds.
Additional analysis such as metabolite ID, Lipid ID, or targeted protein ID/quantitation using peptide SRM/MRMs are also available.
Discovery quantitation of differences in protein amount between samples can be done by in vivo SILAC metabolic labeling or iTRAQ isotope tagging post-harvest (up to 8 separate samples with 500-2500 proteins ID'd and quantitated in single experiments), or by MS identification of spots of interest on 1D/2D gels. For quantitation of lists of pre-determined proteins of interest, peptide SRM (MRM) assays can be developed to allow rapid sequential relative or (with heavy isotope peptide standards) absolute quantitation of proteins of interest. Finally, combined ID and label-free quantitation ("Qual/Quant") analyses can be performed using Data-Independent Analysis (DIA) SWATH methods
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Primary Services Offered:
- Mass Spectrometry analyses and identification of proteins, peptides, oligonucleotides, carbohydrates, small molecules
- 2D LC separations of complex protein and/or peptide mixtures for Proteomics - up to 5000-7000 confident Protein IDs from a single sample
- Protein Expression analysis using iTRAQ/ICAT, SILAC, or 15N methodology - relative quantitation of thousands of proteins across up to 8 samples simulataneously
- SWATH/MSALL Data-Independent Acquisition for simultaneous identification and label-free quantitation of peptides/proteins/lipids/small molecules
- Proteomic Data Analysis using both commercial and in-house created software suites
MS Quantitation using SRM/MRM (Selective/Multiple Reaction Monitoring)
antibody-free accurate quantitation of protein levels in 100's of samples
accurate quantitation of small molecules, hormones, NTPS, drugs, and metabolites (e.g., for pharmacokinetic studies, drug metabolism studies)
accurate quantitation of cytokines, amino acids, etc. from microdialysates
- Protein Expression analysis using 2D Gel-image analysis
- Accurate Quantitation of Cytokines, Insulin, Glucagon, etc. using MSD Sector Imager 2400 Electroluminescent Plate Reader
- Automated spot-cutting, proteolytic digestion, and sample deposition from gels
- Shared equipment for analyzing whole protein sizes and multimeric state
- ABSciex TripleTOF 5600+ mass spec
- ABI/MDS Sciex 5800 MALDI TOF-TOF mass spec
- MDS Sciex 4000 QTrap hybrid ion trap mass spec
- Waters Synapt HDMS hybrid QTOF with Ion Mobility
- ABI Voyager DE-PRO Reflectron MALDI TOF mass spec
- MesoScale Discoveries (MSD) Sector Imager 2400 Electrochemiluminescence Detector/Plate Reader
- Bioinformatics: ProteinPilot and Phenyx advanced protein-MS ID software; Mascot software, Analyst software, MultiQuant, Skyline
- HPLC/UPLC and Gel Separation Systems:
- Eksigent NanoLC-Ultra-2D Plus, cHiPLC Nanoflex
- Waters Acquity UPLC and NanoAcquity 2D nanoLC liquid chromatography systems
- ÄKTA Prime Plus system
- Agilent 1100, Shimadzu LC-AS10, Waters Alliance 2695 & 600E HPLC systems
- Tempo LC-MALDI nanoflow separation & MALDI spotting system
- Beckman-Coulter PF 2D Whole Protein separation system
- 12-gel casting and running apparatus for 2D gels (Ettan IPGPhor II and Dalt 12)
- Gel Imaging and analysis
- BioRad FX Pro Plus phosphorimager, fluorimager (3 laser), and Densitometer with QuantityOne and PDQuest software
- GE/Amersham Typhoon DIGE gel reader with DeCyder software
- Spot-cutting and robotics
- LEAP Technologies/BioMachines 2DiDx Spot-cutting/digestion/spotting robot
- GE/Amersham Ettan Spot Picker
- Tecan EVO Robotics platform (Room C2704)
- Shimadzu CHiP printer
- Wyatt DAWN HELEOS light-scattering detection system for protein sizing
- Biacore 3000 Surface Plasmon Resonance system
In addition to using classic gel spot densitometric (2D Gels) or fluorescence (DIGE Gel) measurements to find proteins whose levels differ between two or more samples, identification and quantitation of protein level changes can be done by using chemical isotope labeling methods. In such methods, one tags all proteins from different samples with identical chemical tags that differ only in their distribution of heavy and light (non-radioactive) isotopes of the same atoms, e.g., 1H vs. 2H, 12C vs 13C, 14N vs. 15N. Where possible, this can be done in living cells or animal models by feeding heavy vs. light isotope versions of various naturally incorporated amino acids (sometimes called SILAC methods); however, for many studies such pre-labeling is not possible. In such cases various chemical isotope incorporation methods are available. We previously performed many analyses with Isotope Coded Affinity Tag (ICAT) methods, and over the past 7 years (2006-2013) we have used Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) methods to perform many hundreds of large scale analyses of complex samples, including human serum samples, animal and plant cell lysates, and cell organelle preps. Such experiments typically identify and quantitate the relative amounts of 500-2500 proteins from 4-8 samples at once (see further details below).
Standard sample separations for iTRAQ experiments done in the Mass Spec Facility (after the iTRAQ reagent labeling step) are identical to those for an LC-MALDI MudPit experiment:
- Separation of the combined 4 or 8 iTRAQ-labeled samples offline into 15 strong cation exchange fractions, using a 4.6 X 250 mm PolySULFOETHYL Aspartamide Strong Cation exchange column (PolyLC, Columbia, MD) with an ammonium formate gradient in 20% acetonitrile. (Details of these protocols can be viewed at "2D LC Procedures Used")
- SpeedVac drying and resuspension in H2O of all fractions (3X) to remove all acetonitrile and ammonium formate.
- Separation of each SCX fraction on a 15 cm Reprosil C18 nanoflow column on an Eksigent ChipLC system, and direct injection into our ABSciex 5600 TripleTOF mass spectrometer (For MALDI analyses, separation of each SCX fraction on an LC-Tempo nanoflow separation and MALDI spotting system, using a Chromolith CapRod column C18 column (150 X 0.1 mm, Merck), into 370 MALDI spots on a stainless steel MALDI target plate, then adding 13 calibration spots to the same target plate).
- Instrument is calibrated with an injection of beta-gal digest standard before each sample injection (For MALDI analyses, update plate calibration and MS/MS default calibration for each plate as it goes into our Applied Biosystems 5800 MALDI TOF TOF mass spectrometer).
- A 250 ms parent scan is acquired, then up to 50 MS/MS spectra over 2.5 seconds, then repeated with the next LC eluant fraction. (For MALDI analyses, acquisition of 400 laser shots for MS spectra from each spot, then data-dependent acquisition of MS/MS spectra for each peptide mass, with the MS/MS spectra taken from the spot containing the largest MS peak representing each of the peptide peaks observed across the entire plate).
- Combination of the MS/MS data from all 15 SCX fractions for a Paragon algorithm search (ProteinPilot 4.5 Software as of Jan 2013, against a concatenated normal and reversed database (SwissProt, NCBI, etc.). This search of a concatenated normal and reversed database allows estimation of the False Discovery Rate (or False Positive Rate) so that one can set the score limits for "positive IDs" to only accept IDs where the local False Discovery Rate estimate of the lowest ranking protein is 5%, and all higher ranking proteins have decreasingly lower probabilities of being false positives (See "Calculating False Discovery Rates" for more information on the importance of using False Discovery Rate estimations and how they are calculated.
The resulting ProteinPilot .group files can be viewed with a Windows XP or later computer by installing a trial version of the ProteinPilot Software from http://www.absciex.com/products/software/proteinpilot-software - once the trial period is over, you can continue to use the software as a viewer for .group files produced by our licensed versions of the software.
Many Small Molecule analyses (polymer analysis, validation of synthesized compounds) can be performed as a walk-up service on our Voyager DE-PRO Reflectron MALDI instrument in room C1734 (after training by Core personnel on the use of the instrument).
More complex analyses requiring MS/MS and MS3 fragmentation, or requiring electrospray-, APCI-, or photo-ionization are performed on one of three mass spec instruments.
Our ABSciex TripleTOF 5600 with a Shimadzu UFLC-XR separation system (C1735), and our Waters Synapt HDMS with Ion Mobility and a Waters Acquity UPLC separation system (C1733), provide strong capacities for small molecule, lipidomic, and metabolomic analyses. Please contact Dr. Dongxiao Sun (email@example.com) to arrange analyses using these instruments.
The MDS/Sciex 4000 QTrap (C1733) includes a PAL CTC temperature-controlled auto sampler, an Agilent 1100 2D LC system, all standard quadrupole and ion trap MS modes, and various enhanced modes for increased sensitivity and selectivity. This instrument includes the possibility of MS^3 analyses, as well as additional ionization modes possibly advantageous for analyses of some types of molecules (APCI or PhotoIonization). Please contact Dongxiao Sun for more information [firstname.lastname@example.org, or (717) 531-0003 ext. 287146]
Some recent publications which used data generated in the Mass Spectrometry Core are listed below. Mass Spec Facility staff authors are in bold:
Wase N, Black P, Stanley B, DiRusso C. (2014) Integrated Quantitative Analysis of Nitrogen Stress Response in Chlamydomonas Reinhardtii Using Metabolite and Protein Profiling. J. Proteome Res., 13(3):1373-1396 DOI: 10.1021/pr400952z. PMID: 24528286
Giardina BJ, Stanley BA, Chiang HL. (2014) Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae. Proteome Sci. 12(1):9. doi: 10.1186/1477-5956-12-9. PMID: 24520859
Olsen KC, Xu Y, Chen G, Hajnal, A, Lynch CJ. (2014) Alloisoleucine differentiates the branched-chain aminoacidemia of obese Zucker and diet-induced obesity (DIO) rats. Nature Obesity 22(5):1212-5. doi:10.1002/oby.20691 PMID: 24415721
Chung SH, Rosa C, Scully ED, Peiffer M, Tooker JF, Hoover K, Luthe DS, Felton GW. (2013) Herbivore exploits orally secreted bacteria to suppress plant defenses. Proc Natl Acad Sci U S A. 110(39):15728-33. PMID: 24019469
Bansal R, Helmus RA, Stanley BA, Zhu J, Liermann LJ, Brantley SL, Tien M (2013) Survival During Long Term Starvation: Global Proteomics Analysis of Geobacter sulfurreducens under Prolonged Electron Acceptor Limitation. J. Proteome Res., • DOI: 10.1021/pr400266m • Publication Date (Web): 27 Aug 2013
Skibinski C.G., Thompson, H.J. Das, A. Manni, A., Bortner, Stanley, A., Stanley, B., El-Bayoumy, K. (2013) Proteomic Changes Induced by Effective Chemopreventive Ratios of n-3:n-6 Fatty Acids and Tamoxifen against MNU-Induced Mammary Cancer in the Rat. Cancer Prevention Research. published 23 July 2013, 10.1158/1940-6207.CAPR-13-0152
Olsen KC, Chen G, Lynch CJ. (2013) Quantification of Branched-Chain Ketoacids in Tissue by Ultra Fast Liquid Chromatography-Mass Spectrometry Analytical Biochemistry (2013) Aug 15;439(2):116-22. doi: 10.1016/j.ab.2013.05.002. Epub 2013 May 14. PMID: 23684523
Dennis MD, Shenberger JS, Stanley BA, Kimball SR, Jefferson LS.(2013) Hyperglycemia Mediates a Shift from Cap-Dependent to Cap-Independent Translation via a 4E-BP1 Dependent Mechanism. Diabetes. 2013 Feb 22. [Epub ahead of print] PMID: 23434932
Barth BM, Shanmugavelandy SS, Kaiser JM, McGovern C, Altinoglu EI, Haakenson JK, Hengst JA, Gilius EL, Knupp SA, Fox TE, Smith JP, Ritty TM, Adair JH, Kester M. (2013) PhotoImmunoNanoTherapy Reveals an Anticancer Role for Sphingosine Kinase 2 and Dihydrosphingosine-1-Phosphate. ACS Nano. 2013 Feb 14. [Epub ahead of print]
Das A., Bortner Jr. JD, Aliaga CA, Baker A, Stanley A, Stanley BA, Kaag M, Richie Jr. JP, El-Bayoumy K. (2013) Changes in Proteomic Profiles in Different Prostate Lobes of Male Rats throughout Growth and Development and Aging Stages of the Life Span. The Prostate 73(4):363-75. PMID: 22911278
Kaiser JM, Imai H, Haakenson JK, Brucklacher RM, Fox TE, Shanmugavelandy SS, Unrath KA, Pedersen MM, Dai P, Freeman WM, Bronson SK, Gardner TW, Kester M. (2013) Nanoliposomal minocycline for ocular drug delivery. Nanomedicine. 2013 Jan;9(1):130-40. doi: 10.1016/j.nano.2012.03.004. Epub 2012 Mar 28.
Watters RJ, Fox TE, Tan SF, Shanmugavelandy S, Choby JE, Broeg K, Liao J, Kester M, Cabot MC, Loughran TP, Liu X. (2013) Targeting glucosylceramide synthase synergizes with C(6)-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia. Leuk Lymphoma. 2013 Jun;54(6):1288-96. (2012 Dec 31. [Epub ahead of print])
Fox TE, Young MM, Pedersen MM, Han X, Gardner TW, Kester M. (2012) Diabetes diminishes phosphatidic acid in the retina: a putative mediator for reduced mTOR signaling and increased neuronal cell death. Invest Ophthalmol Vis Sci. 2012 Oct 19;53(11):7257-67. doi: 10.1167/iovs.11-7626.
Madhunapantula SV, Hengst J, Gowda R, Fox TE, Yun JK, Robertson GP. (2012) Targeting sphingosine kinase-1 to inhibit melanoma. Pigment Cell Melanoma Res. 2012 Mar;25(2):259-74. doi: 10.1111/j.1755-148X.2012.00970.x.
Lancaster, T.L., Jefferson, S.J., Hunter, J.C., Lopez, V.U., Van Eyk, J., Lakatta, E.G. and Korzick, D.H. (2012) Quantitative Proteomic Analysis Reveals Novel Mitochondrial Targets of Estrogen Deficiency in the Aged Female Rat Heart. Physiol Genomics 44(20): 957-969. PMID: 22930739
Moon, MS, McDevitt, E, Stanley, BA, Krzeminski, J, Amin, S, Aliaga, C, Miller, T, Isom, H. (2012) Elevated Hepatic Iron Activates NF-E2-Related Factor 2 (NRF2) Regulated Pathway in a Dietary Iron Overload Mouse Model Toxicol. Sci. 129(1): p. 74-85
Liu, X, Liu, D, Qian, D, Dai, J, An, Y, Jiang, S, Stanley, BA, Yang, J, Wang, B, Liu, X, Liu, DX. (2012) . 287(23):19599-609. PMID: 22528486
Giardina, BJ, Stanley, BA, Chiang, H-L. (2012) Comparative Proteomic Analysis of Transition of Saccharomyces cerevisiae from Glucose-Deficient Medium to Glucose-Rich Medium. Proteome Science 10(1): 40. PMID: 22691627
Kuhns EH, Seidl-Adams I, Tumlinson J.H. (2012) A lepidopteran aminoacylase (L-ACY-1) in Heliothis virescens (Lepidoptera:Noctuidae) gut lumen hydrolyzes fatty acid-amino acid conjugates, elicitors of plant defense Insect Biology & Molecular Biology 42(1): 32-40
Fox, TE, Bewley, MC, Unrath, KA, Pedersen, MM, Anderson, RA, Jung, DY, Jefferson, LS, Kim, JK, Bronson, SK, Flanagan, JM, Kester, M. (2011) Circulating sphingolipid biomarkers in models of type 1 diabetes J Lipid Res. 52(3): 509–517. PMCID: PMC3035687
Hankins JL, Fox TE, Barth BM, Unrath KA, Kester M. (2011) Exogenous ceramide-1-phosphate reduces lipopolysaccharide (LPS)-mediated cytokine expression. J Biol Chem. 2011 Dec 30;286(52):44357-66. doi: 10.1074/jbc.M111.264010. Epub 2011 Nov 7.
Jiang Y, DiVittore NA, Kaiser JM, Shanmugavelandy SS, Fritz JL, Heakal Y, Tagaram HR, Cheng H, Cabot MC, Staveley-O'Carroll KF, Tran MA, Fox TE, Barth BM, Kester M. (2011) Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer. Cancer Biol Ther. 2011 Oct 1;12(7):574-85. Epub 2011 Oct 1.
O'Neill SM, Yun JK, Fox TE, Kester M. (2011) Transcriptional regulation of the human neutral ceramidase gene. Arch Biochem Biophys. 2011 Jul;511(1-2):21-30. doi: 10.1016/j.abb.2011.04.012. Epub 2011 Apr 22.
Lancaster TS, Jefferson SJ, Korzick DH (2011) Local delivery of a PKC-epsilon-activating peptide limits ischemia reperfusion injury in the aged female rat heart. Am J Physiol Regulatory Integrative Comp Physiol. 301(5): R1242-R1249
Wilson RT, Roff A, Dai P, Fortugno T, Douds J, Chen G, Grove GL, Nikiforova SO, Barnholtz-Sloan J, Frudakis T, Chinchilli V, Hartmann T, Demers L, Shriver MD, Canfield V, Cheng K (2011) Genetic ancestry, skin reflectance and pigmentation genotypes in association with serum vitamin D metabolite balance. Horm Mol Biol Clin Invest 7(1):279-293 DOI 10.1515/HMBCI.2011.021
Losiewicz MK, Fort, PE. (2011) Diabetes impairs the neuroprotective properties of retinal alpha-crystallins. Invest. Ophthalmol. Vis. Sci. published 5 April 10.1167/iovs.10-6931
Fogle RL, Hollenbeak CS, Stanley BA, Vary TC, Kimball SR, Lynch CJ. (2011) Functional proteomic analysis reveals sex-dependent differences in structural and energy-producing myocardial proteins in rat model of alcoholic cardiomyopathy. Physiol Genomics 43(7): p. 346-356 PMID: 21245415
Bortner JD, Richie JP, Das A, Liao J, Umstead TM, Stanley A, Stanley BA, Belani CP, El-Bayoumy K. (2011) Proteomic Profiling of Human Plasma by iTRAQ Reveals Down-Regulation of ITI-HC3 and VDBP by Cigarette Smoking. J Proteome Res; Mar 4;10(3):1151-9. PMID: 21186835
VanGuilder HD, Bixler GV, Kutzler L, Brucklacher RM, Bronson SK, Kimball SR, Freeman WM. (2011) Multi-modal proteomic analysis of retinal protein expression alterations in a rat model of diabetic retinopathy. PLoS One. Jan 13;6(1):e16271
Desai, D., Salli, U., Vrana, K. E and Amin, S. (2010). SelSA, selenium analogs of SAHA as potent histone deacetylase inhibitors. Biorganic and Med Chem Letters 20, 2044-2047. PMID: 20167479
Desai, D., Madhunapantula, S.V., Gowdahalli, K., Sharma, A., Chandagaludoreswany, R., El-Bayoumy, K., Robertson, G.P. and Amin, S. (2010). Synthesis and characterization of a novel iNOS/Akt inhibitor Se,Se'-1,4-phenylenebis(1,2-ethanediyl)bisisoselenourea (PBISe)-against colon cancer. Biorganic and Med Chem Letters 20, 2038-2043. PMID: 20153642
Fogle RL, Lynch CJ, Palopoli M, Deiter G, Stanley BA, Vary TC. (2010) Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression. Alcohol Clin Exp Res. (2010) Jul;34(7):1226-34 PMID: 20477769
Moura, AA, Souza CE, Stanley BA, Chapman DA, Killian GJ. (2010) Proteomics of cauda epididymal fluid from mature Holstein bulls. J Proteomics. Sep 10;73(10):2006-20 PMID: 20601273
Ali M, Umstead TM, Haque R, Mikerov AN, Freeman WM, Floros J, Phelps DS. (2010) Differences in the BAL proteome after Klebsiella pneumoniae infection in wild type and SP-A-/- mice. Proteome Sci. 2010 Jun 17;8:34. PMID: 20565803
Umstead TM, Lu CJ, Freeman WM, Myers JL, Clark JB, Thomas NJ, Chinchilli VM, Vrana KE, Undar A, Phelps DS. (2010) Dual-platform proteomics study of plasma biomarkers in pediatric patients undergoing cardiopulmonary bypass. Pediatr Res. 2010 Jun;67(6):641-9. PMID: 20308938
Khanna MR, Stanley BA, Thomas GH. (2010) Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane. BMC Genomics. 2010 May 12;11(1):302. PMID: 20462449.
Winter JN, Fox TE, Kester M, Jefferson LS, Kimball SR (2010) Phosphatidic acid mediates activation of mTORC1 through the ERK signaling pathway Am J Physiol Cell Physiol 299:C335-C344, 2010 First published April 2010; doi:10.1152/ajpcell.00039.2010
VanGuilder HD, Yan H, Farley JA, Sonntag WE, Freeman WM. (2010) Aging alters the expression of neurotransmission-regulating proteins in the hippocampal synaptoproteome. J Neurochem. 2010 Jun;113(6):1577-88. Epub 2010 Mar 31 PMID: 20374424
Zhao Z, Stanley BA, Zhang W, Assmann SM (2010) ABA-regulated G protein signaling in Arabidopsis guard cells: a proteomic perspective. J Proteome Res. 2010 5;9(4):1637-47. PMID: 20166762
Barthéléry, M., Jaishankar, A., Salli, U., and Vrana, K. E. (2009). Reptin52 expression during in vitro neural differentiation of human embryonic stem cells. Neurosci Lett 452, 47-51. PMID: 19444951
Barthéléry M, Salli U, Freeman WM, Vrana KE. (2009) 2-DIGE identification of differentially expressed hNRNPs and transcription factors during neural differentiation of human ES cells. Proteomics: Clinical Applications 3:505-514, 2009
Bortner JD Jr, Das A, Umstead TM, Freeman WM, Somiari R, Aliaga C, Phelps DS, El-Bayoumy K. (2009) Down-regulation of 14-3-3 isoforms and annexin A5 proteins in lung adenocarcinoma induced by the tobacco-specific nitrosamine NNK in the A/J mouse revealed by proteomic analysis. J Proteome Res. 2009 Aug;8(8):4050-61. PMID: 19563208
Boyiri, T., Somiari, R. I., Russell, S., Aliaga, C., and El-Bayoumy, K. (2009). Proteomics of rat prostate lobes treated with 2-N-hydroxylamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 5alpha-dihydrotestosterone, individually and in combination. Int J Oncol 35, 559-567. PMID: 19639176
Crown PL, Hurst WJ. (2009) Evidence of cacao use in the Prehispanic American Southwest. Proc Natl Acad Sci U S A. 2009 Feb 2. 106(7): 2110-2113 PMID: 19188605
Culnan DM, Cooney RN, Stanley B, Lynch CJ. (2009) Apolipoprotein A-IV, a Putative Satiety/Antiatherogenic Factor, Rises After Gastric Bypass. Obesity (Silver Spring). 2009 Jan;17(1):46-52. PMID: 18948973
Wijesinghe DS, Allegood JC, Gentile LB, Fox TE, Kester M, Chalfant CE. (2009) Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels. J Lipid Res. 2010 Mar;51(3):641-51. doi: 10.1194/jlr.D000430. Epub 2009 Aug 4.
Das, A., Bortner, J., Desai, D., Amin, S., and El-Bayoumy, K. (2009). The selenium analog of the chemopreventive compound S,S'-(1,4-phenylenebis[1,2-ethanediyl])bisisothiourea is a remarkable inducer of apoptosis and inhibitor of cell growth in human non-small cell lung cancer. Chem Biol Interact 180, 158-164. PMID: 19497413
Feng B, Li L, Zhou X, Stanley B, Ma H. (2009) Analysis of the Arabidopsis Floral Proteome: Detection of over 2,000 Proteins and Evidence for Posttranslational Modifications. J Integr Plant Biol. 2009 Feb;51(2):207-23. PMID: 19200160
Freeman WM, VanGuilder HD, Bennett C, Sonntag WE. (2009) Cognitive performance and age related changes in the hippocampal proteome. Neuroscience 159:183-9, 2009. PMID:
Haque R, Umstead TM, Freeman WM, Floros J, Phelps DS. (2009) The impact of surfactant protein-A on ozone-induced changes in the mouse broncoalveolar lavage proteome. Proteome Sci. 7:12, 2009
Hengst JA, Guilford JM, Fox TE, Wang X, Conroy EJ, Yun JK. (2009) Sphingosine kinase 1 localized to the plasma membrane lipid raft microdomain overcomes serum deprivation induced growth inhibition. Arch Biochem Biophys. 492(1-2):62-73, 2009. PMID: 19782042.
Hurst WJ, Stanley B, Glinski JA, Davey M, Payne MJ and Stuart DA (2009) Characterization of Primary Standards for Use in the HPLC Analysis of the Procyanidin Content of Cocoa and Chocolate Containing Products. Molecules 14(10): 4136-4146, 2009 doi:10.3390/molecules14104136
Jaishankar A, Barthéléry M, Freeman WM, Salli U, Vrana KE. (2009) Human Embryonic and Mesenchymal Stem Cells Express Different Nuclear Proteomes. Stem Cells Develop. 18:793-802, 2009
Lee, J. I., Nian, H., Cooper, A. J., Sinha, R., Dai, J., Bisson, W. H., Dashwood, R. H., and Pinto, J. T. (2009). Alpha-keto acid metabolites of naturally occurring organoselenium compounds as inhibitors of histone deacetylase in human prostate cancer cells. Cancer Prev Res (Phila Pa) 2, 683-693. PMID: 19584079
Lull ME, Erwin MS, Patel KM, Morgan D, Roberts DCS, Vrana KE, Freeman WM. (2009) Persistent proteomic alterations in medial prefrontal cortex with abstinence from cocaine self-administration. Proteomics: Clinical Applications 3:462-472, 2009.
Nowak, M. G., Sudol, M., Lee, N. E., Konsavage, W. M., Jr., and Katzman, M. (2009). Identifying amino acid residues that contribute to the cellular-DNA binding site on retroviral integrase. Virology 389, 141-148. PMID: 19447461
Nyland, S. B., and Loughran, T. P. (2009). Putting HIV-1 apoptosis mediators to work against cancer: A killer job, with benefits. Cancer Biol Ther 8, 188-191. PMID: 19182513
Popova, E. Y., Krauss, S. W., Short, S. A., Lee, G., Villalobos, J., Etzell, J., Koury, M. J., Ney, P. A., Chasis, J. A., and Grigoryev, S. A. (2009). Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation. Chromosome Res 17, 47-64. PMID: 19172406
Purdy, JG, Flanagan JM, I. J. Ropson IJ, and Craven, R. C. (2009) Retroviral capsid assembly: a role for the CA dimer in initiation. J. Mol. Biol. 389(2):438-51, 2009. PMID: 19361521
Sharma, A., Sharma, A. K., Madhunapantula, S. V., Desai, D., Huh, S. J., Mosca, P., Amin, S., and Robertson, G. P. (2009). Targeting Akt3 Signaling in Malignant Melanoma Using Isoselenocyanates. Clin Cancer Res 15, 1674-1685. PMID: 19208796
Sundstrom JM, Tash BR, Murakami T, Flanagan JM, Bewley MC, Stanley BA, Gonsar KB, Antonetti DA (2009) Identification and Analysis of Occludin Phosphosites: A Combined Mass Spectrometry and Bioinformatics Approach. J Proteome Res. 8(2):808-817 PMID: 19125584
Sundstrom JM, Sundstrom CJ, Sundstrom SA, Fort PE, Rauscher RL, Gardner TW, Antonetti DA. (2009) Phosphorylation Site Mapping of Endogenous Proteins: A Combined MS and Bioinformatics Approach. J Proteome Res. Feb 6;8(2):798-807. PMID: 19125583
Umstead TM, Freeman WM, Phelps DS. (2009) Age-related changes in the expression and oxidation of bronchoalveolar lavage proteins in the rat. AJP: Lung Cellular and Molecular Physiology 296:L14-29, 2009
Zhao Z, Zhang W, Stanley BA, Assmann SM. (2008) Functional Proteomics of Arabidopsis thaliana Guard Cells Uncovers New Stomatal Signaling Pathways. Plant Cell. 2008 Dec;20(12):3210-26. PMID: 19114538
O'Neill SM, Olympia DK, Fox TE, Brown JT, Stover TC, Houck KL, Wilson R, Waybill P, Kozak M, Levison SW, Weber N, Karavodin LM, Kester M. (2008) C(6)-Ceramide-Coated Catheters Promote Re-Endothelialization of Stretch-Injured Arteries. Vasc Dis Prev. 2008 Aug 1;5(3):200-210.
Geguchadze RN, Coe C, Lubach GR, Clardy TW, Beard JL, Connor JR. (2008) CSF Proteomic Analysis Reveals Persistent Iron Deficiency-induced Alterations In Non-human Primate Infants J Neurochem. 2008 Apr;105(1):127-36.
Barthéléry, M., Salli, U., and Vrana, K. E. (2008). Enhanced nuclear proteomics. Proteomics 8, 1832-1838. PMID: 18384104
Chen KM, Spratt TE, Stanley BA, De Cotiis DA, Bewley MC, Flanagan JM, Desai D, Das A, Fiala ES, Amin S, El-Bayoumy K. (2007) Inhibition of nuclear factor-kappaB DNA binding by organoselenocyanates through covalent modification of the p50 subunit. Cancer Res. 67(21):10475-83. PMID: 17974991
Chang SI, El-Bayoumy K, Sinha I, Trushin N, Stanley BA, Pittman B, Prokopczyk B. (2007) 4-(Methylnitrosamino)-I-(3-Pyridyl)-1-Butanone Enhances the Expression of Apolipoprotein A-I and Clara Cell 17-kDa Protein in the Lung Proteomes of Rats Fed a Corn Oil Diet but not a Fish Oil Diet. Cancer Epidemiol Biomarkers Prev 16(2): 228-35. PMID: 17301254
Fort PE, Freeman WM, Losiewicz MK, Singh RS, Gardner TW. (2009) The retinal proteome in experimental diabetic retinopathy: Up-regulation of crystallins and reversal by systemic and periocular insulin. Mol Cell Proteomics. Apr;8(4):767-79 . PMID: 19049959
Kester, M., Heakal, Y., Fox, T., Sharma, A., Robertson, G. P., Morgan, T. T., Altinoglu, E. I., Tabakovic, A., Parette, M. R., Rouse, S. M., et al. (2008). Calcium phosphate nanocomposite particles for in vitro imaging and encapsulated chemotherapeutic drug delivery to cancer cells. Nano Lett 8, 4116-4121. PMID: 19367878
Konsavage, W. M., Jr., Sudol, M., and Katzman, M. (2008). Effects of varying the spacing within the D,D-35-E motif in the catalytic region of retroviral integrase. Virology 379, 223-233. PMID: 18687451
Lin, L., and Hu, J. (2008). Inhibition of hepadnavirus reverse transcriptase-epsilon RNA interaction by porphyrin compounds. J Virol 82, 2305-2312. PMID: 18094191
Lin, L., Wan, F., and Hu, J. (2008). Functional and Structural dynamics of Hepadnavirus Reverse Transcriptase During Protein-Primed Initiation of Reverse Transcription: Effects of Metal Ions. J Virol. 82, 5703-14. PMID: 18400846
Madhunapantula, S. V., Desai, D., Sharma, A., Huh, S. J., Amin, S., and Robertson, G. P. (2008). PBISe, a novel selenium-containing drug for the treatment of malignant melanoma. Mol Cancer Ther 7, 1297-1308. PMID: 18483317
Purdy JG, Flanagan JM, Ropson IJ, Rennoll-Bankert KE, Craven RC. (2008) Critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly. J Virol. 2008 Jun;82(12):5951-61. PMID: 18400856
Lull ME, Carkaci-Salli N, Freeman WM, Myers JL, Midgley FM, Thomas NJ, Kimatian SJ, Vrana KE, Ündar A. (2008) Plasma proteomic biomarkers in pediatric cardiac patients undergoing cardiopulmonary bypass procedures. J. Pediatric Res. 2008; 63:638-644. PMID: 18317239
Salli, U., Fox, T. E., Carkaci-Salli, N., Sharma, A., Robertson, G. P., Kester, M., and Vrana, K. (2008). Propagation of undifferentiated human embryonic stem cells with nano-liposomal ceramide. Stem Cells Dev 18, 55-65. PMID: 18393629
Sharma, A. K., Sharma, A., Desai, D., Madhunapantula, S. V., Huh, S. J., Robertson, G. P., and Amin, S. (2008). Synthesis and anticancer activity comparison of phenylalkyl isoselenocyanates with corresponding naturally occurring and synthetic isothiocyanates. J Med Chem 51, 7820-7826. PMID: 19053750
Stover, T. C., Kim, Y. S., Lowe, T. L., and Kester, M. (2008). Thermoresponsive and biodegradable linear-dendritic nanoparticles for targeted and sustained release of a pro-apoptotic drug. Biomaterials 29, 359-369. PMID: 17964645
Zolnik, B. S., Stern, S. T., Kaiser, J. M., Heakal, Y., Clogston, J. D., Kester, M., and McNeil, S. E. (2008). Rapid Distribution of Liposomal Short-Chain Ceramide In Vitro and In Vivo. Drug Metab Dispos 36, 1709-1715. PMID: 18490436
Consultations, recommendations for reagents and procedures, and instructions are all freely available, please contact Dr. Bruce Stanley by email at email@example.com, or by telephone at (717) 531-5329.
- No charge for initial consultations and feasibility discussions, nor for training on the Voyager DE-PROMALDI-TOF self-service instrument.
- Self-service (Voyager DE-PRO only): $25 per hour
- 5600 TripleTOF, Synapt, 4000 QTrap: Method development, set-up, run time and data analysis : $65.00 per hour (as of 8/15/12)
Analyses/Syntheses performed by facility staff
- Peptide Synthesis
- 0.1 mmol half-scale synthesis - $20 per amino acid
- 0.2 mmol full-scale synthesis - $30 per amino acid
- Mass Spec validation of peptide MW costs an additional $12
- Simple clean, desalted samples such as purified proteins or in-gel digestions of single spots can be analyzed on our DE-PRO MALDI-TOF or 5800 MALDI TOF-TOF by Mass Spec Core personnel:
- mass within 0.05% - $10 per sample
- mass within 0.015% using external/internal calibrants - $12 per sample
- Removal of interfering salts or detergents if necessary (ZipTips) is an additional $15 charge per sample - recommended for all gel spot digests
- Trypsin or other proteolytic digestions prepared by Mass Spec Core personnel:
- $30 set-up fee plus $30 per sample digested - includes ZipTip cleanup/desalting and plate spotting
- MS analysis of previously digested and desalted simple sample (e.g., gel spot), data-dependent MS/MS analysis, and protein identification (computer analysis of spectra obtained to identify protein): $12. If you provide us with already spotted samples on a usable MALDI target plate, the fee is $10 per spot.
- EXAMPLE 1: If you send us 22 gel slices for us to trypsin digest and analyze, your cost will be $30 Trypsin digest set-up fee PLUS (22 samples X $30 per trypsin digest/ZipTip cleanup) PLUS (22 samples X $12 per sample Mass Spec and database analysis) = $954.
- EXAMPLE 2: If you send us the same 22 samples already digested, but needing ZipTip cleanup/spotting and MS/MS analysis, your cost will be (22 samples X $15 ZipTip cleanup/spotting) PLUS (22 samples X $12 Mass Spec analysis) = $594
- EXAMPLE 3: If you send us the same 22 digested, ZipTip cleaned samples already spotted on a MALDI target plate, your cost will be 22 samples X $10 Mass Spec and Database analysis = $220
- Single dimension C18 nanoflow-LC separations and MS plus MS/MS analysis of a single more complex sample (e.g., multiple proteins) are performed for $130 (necessary for example to maximize sequence coverage when searching for post-translational modifications, takes the place of ZipTip cleanup and MS MS/MS analysis above)
- Other Staff time is charged at $60 per hour
- More complex samples and analyses such as user-provided tryptic digests for MudPit or iTRAQ analyses, or exhaustive post-translational modification analyses, are between $130 and $1250 per sample depending on the extent of additional processing and staff analysis time needed. For example, MudPit and iTRAQ or ICAT labeled samples require two-dimensional chromatographic separation followed by MS and MS/MS analysis and database searching - if you provide us a digested, iTRAQ labeled sample (see iTraq Protocols), the cost of the 2D-LC and MS/MS analyses is $1250. Please discuss your specific project and experimental goals with Dr. Stanley to determine the analyses and separations needed and the price for these more complex services.
- We purchase iTraq reagents in bulk and provide them to researchers at cost (this service is provided only to researchers who are having their analyses done here). A set of 8-Plex reagents is $550, and a set of 4-plex reagents is $275 - if the reagents need to be shipped to you, the cost is an additional $40-$60 per shipment.
Consultations, recommendations for reagents and procedures, and instructions are all freely available. Please contact Dr. Bruce Stanley by email at firstname.lastname@example.org, or by telephone at 717-531-5329.
- Use of Protease inhibitors during Proteomic Sample Prep
- In-Gel Digestion Protocols
- Solution Digestion Protocols
- iTRAQ Sample Prep Protocol
- ElectroBlot Protocol for Edman Sequencing
- iTRAQ Mass Spec Sample Submission Form
- Non-iTRAQ Protein Mass Spec Sample Submission Form (MudPit, gel-spot digests, etc.)
- Small Molecule Mass Spec Sample Submission Form (Metabolites, Lipids, etc.)
Using the Proteus & Geneus LIMS System for Sample Submission and Data Retrieval
- Getting a Proteus/Geneus LIMS account - see instructions below.
- Click HERE to log on to the Proteus LabLink System for Data Retrieval or Sample Submission
- Click HERE to log on to the Geneus LabLink System for Data Retrieval or Sample Submission
Instructions for obtaining a LIMS account:
Signing Up for a Proteus/Geneus LIMS LabLink Account (download PDF of instructions below)
Note that Proteus LabLink & Geneus LabLink are very similar, but SEPARATE LIMS systems - an account on one system does NOT allow you access to the other system, you must have TWO accounts, one for each system:
- Proteus serves the Mass Spec, Flow Cytometry, and Microscopy Imaging Cores
- Geneus serves the Functional Genomics and DNA Sequencing Cores
For your username fill in your Penn State Access Account ID (e.g., bas12). (For external customers, before you can request a LabLink account you will need a Friends of Penn State ID, which you can apply for by following the "Create an Account" link and instructions at https://fps.psu.edu/ )
For your username fill in your Penn State Access Account ID (e.g., bas12). (For external customers, before you can request a LabLink account you will need a Friends of Penn State ID, which you can apply for by following the "Create an Account" link and instructions at https://fps.psu.edu/ )
3). When your account request has been approved by IT (usually takes 2-3 days), you will receive an email with your username and temporary password, which you can then use to login to Submit Sample/Analysis Requests or Retrieve Data as follows:
a). For Proteus LabLink, log in at: https://proteuslablink.med.psu.edu/lablink/Login.do
b). For Geneus LabLink, log in at: https://geneuslablink.med.psu.edu/lablink/Login.do
4). The first time you log on to either system, please change your password to something you can remember.
For suggestions or comments about the Penn State Hershey Core Research Facilities website, please email Dr. Bruce A. Stanley at email@example.com, or fill in an anonymous Feedback Form.
| Director |
Bruce Stanley, M.S., PhD (Cornell University)
| Proteomics Lab Manager |
Anne Stanley, D.U.T. (IUT de Strasbourg)
Methods Development, Small Molecule & Amino Acids Analyses
Dongxiao Sun, Ph.D (The University of Hong Kong)
Instructor, Department of Pharmacology
Penn State Cancer Institute
Penn State Cancer Institute
Room C1733 lab
Office Phone (717)531-0003 x280575
Lab Phone (717)531-4055
Ship samples to:Mass Spec Core
Penn State College of Medicine
500 University Dr
Hershey, PA 17033