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Mass Spectrometry - An old tool with many applications
in biomedical research
The University
of Louisville Mass Spectrometry Core Laboratory
promotes the use
of these research tools , and serves as a
resource for University
researchers.
Dr.
Bill Pierce - Laboratory Director
Dr.
Jian Cai - Technical Director
Mr.
Ned B. Smith - Laboratory Manager
How we apply these techniques
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Click
here for a Sample Submission Form
Here is a general price list (price list is linked) Here is a discounted price list for Investigators from the State of Kentucky (price list is linked)
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Ionization Techniques
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m/z Analysis Techniques
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| Publications | Collaborators / Users and their Applications |
| Instrumentation
Micromass Quattro LC [ESI-MS-MS ]
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Useful applications of mass spectrometry -
Through the 1980s and 1990s, most of our focus
was on applications of mass spectrometry to the analyses of small molecules
[<500 Da]. Recently our focus has turned to Biomolecular Mass
Spectrometry. We participate in the University of Louisville Mass
Spectrometry Core Laboratory. We enjoy collaborative research and
we perform fee-for-service analyses.
Here are some of our publications and presentations that include applications of mass spectrometry
Singh S, Powell DW, Rane MJ, Millard TH, Trent JO, Pierce WM, Klein JB, Machesky LM, McLeish KR Identification of the p16-Arc subunit of the Arp2/3 complex as a substrate of MAPK-activated protein kinase-2 by proteomic analysis. J Biol Chem. 2003 Jun 26
Klein JB, Gozal D, Pierce WM, Thongboonkerd V, Scherzer JA, Sachleben LR, Guo SZ, Cai J, Gozal E. Proteomic identification of a novel protein regulated in CA1 and CA3 hippocampal regions during intermittent hypoxia. Respir Physiol Neurobiol. 2003 Jul 16;136(2-3):91-103.
Powell DW, Rane MJ, Joughin BA, Kalmukova R, Hong JH, Tidor B, Dean WL, Pierce WM, Klein JB, Yaffe MB, McLeish KR. Proteomic Identification of 14-3-3zeta as a Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Substrate: Role in Dimer Formation and Ligand Binding. Mol Cell Biol. 2003 Aug 1;23(15):5376-5387.
Thongboonkerd, V., J Luengpailan, J Cao, WM Pierce, Jr., J Cai, JB Klein and RJ Doyle. Fluoride exposure attenuates expression of Streptococcus pyogenes virulence factors. J. Biol. Chem. 277:16599-16605, 2002
Ping, P, C Song, J Zhang, Y Guo, X Cao, RC Li, W Wu, TM Vondriska, JM Pass, XL Tang WM Pierce Jr., and R Bolli. Formation of protein kinase C(epsilon)-Lck signaling module confers cardioprotection. J Clin Invest. 109:499-507, 2002.
Molestina, RE, JB Klein, RD Miller, WM Pierce Jr., JA Ramirez and JT Summersgill. Proteomic analysis of differentially expressed Chlamydia Pneumoniae genes during persistent infection of Hep-2 Cells. Infection and Immunity 70:2976-2981, 2002.
Castegna,A M Aksenov, M Aksenova, V Thongboonkerd, JB Klein, WM Pierce, R Booze, WR Markesberry and DA Butterfield. Proteomic identification of Oxidatively Modified Proteins in Alzheimer's disease Brain. Part I: Creatine Kinase BB, Glutamine Synthase, and Ubiquitin Carboxy-terminal Hydrolase L-1. Free Radical Biol Med 2002 Aug 15;33(4):562
Arthur, John M., Visith Thongboonkerd, Janice A. Scherzer, Jian Cai, William M. Pierce and Jon B. Klein. Differential expression of proteins in renal cortex and medulla: A proteomic approach. Kidney Int. 2002 Oct;62(4):1314-21.
Castegna, A., M. Aksenov, V. Thongboonkerd, JB Klein, WM Pierce Jr., R. Booze, WR Markesbery and DA Butterfield. Proteomic Identification of Oxidatively Modified Proteins in Altzheimer’s disease brian. Part II: hihydropyrimidinase-related protein 2, enolase and heat shock cognate 71. J Neurochem. 2002 Sep;82(6):1524-32.
Gozal, E., D Gozal, WM Pierce, V Thongboonkerd, JA Scherzer, LR Sachleben Jr., S-Z. Guo, J Cai, and JB. Klein. Proteomic Analysis of CA1 and CA3 Regions of Rat Hippocampus and Differential Susceptibility to Intermittent Hypoxia. J Neurochem. 2002 Oct;83(2):331-45.
Thongboonkerd, V., E. Gozal, LR Sachleben, Jr., JM Arthur, WM Pierce Jr., J. Cai, J. Chao and JB Klein. Proteomic Analysis Reveals Alterations in the Renal Kallikrein Pathway During Hypoxia-Induced Hypertension. J Biol Chem. 2002 Sep 20;277(38):34708-16
Feng, W, J Cai, WM Pierce, Jr., and Z-H Song. Expression of functional CB2 cannabinoid receptor in Pichia pastoris for purification and mass spectrometric characterization. Protein Expr Purif. 2002 Dec;26(3):496-505
Klein JB, Pierce WM, Scherzer JA, Cai J, Sachleben LR, Gozal D, Gozal E. Proteomic analysis of CA1 and CA3 regions of hippocampus following brief exposures to continuous hypoxia reveals disparities in antioxidant expression. Presented at: 32nd Annual Meeting of the Society for the Neurosciences, 2-5 November, 2002, Orlando, FL, Abs. # 577.7.
Castegna A, Aksenov M, Aksenova M, Booze R, Markesberry W, Butterfield DA, Thongboonkerd V, Klein J, Pierce W. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain: Creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Neurobiology of Aging 23 (1): 1861 Suppl. 1 JUL-AUG 2002.
Adeagbo AS, Joshua KG, Prough RA, Pierce WM, Awe SO, Falkner KC. Endothelium-derived hyperpolarizing factor mediated vasodilation during DOCA-salt induced hypertension. Hypertension 40 (3): P49 SEP 2002.
Thongboonkerd V, McLeish KR, Epstein PN, Pierce WM, Klein JB. Proteomic analysis of hypoinsulinemic diabetic nephropathy. Journal of the American Society of Nephrology 13: 120A-121A Suppl. S SEP 2002.
Expression of CB2 cannabinoid receptor in Pichia pastoris for purification and characterization. W. Feng, J. Cai, W. Pierce and Z. H. Song, XIVth World Congress of Pharmacology, 2002.
Purification and Characterization of CB2 Cannabinoid Receptor Expressed in Pichia Pastoris. Z. H. Song, J. Cai, W. Pierce, and W. Feng, International Cannabinoid Research Society Conference, 2002.
Nerland, D.E., J. Cai, W.M. Pierce, Jr. and F.W. Benz. Covalent binding of acrylonitrile to specific rat liver glutathione-S-transferases in vivo. In press Chem. Res. Toxicol., 2001
Fitzpatrick, J.L., S.L. Ripp, N.B. Smith, W.M. Pierce, Jr. and R.A. Prough. Metabolism of DHEA by Cytochromes P450 in Rodent and Human Liver Microsomal Fractions. In press Arch. Biochem. Biophys. XXX:XX-XX, 2001 .
Ping, P, J Zhang, WM Pierce, Jr. and R. Bolli. Functional Proteomic Analysis of PKC? Signaling Complexes Assocaited with Cardioprotection. Circ Res. Jan 19;88(1):59-62, 2001 .
Madhavi J. Rane, Patricia Y. Coxon, David W. Powell, Rose Webster, Jon B. Klein, Peipei Ping, William Pierce, and Kenneth R. McLeish p38 kinase-dependent MAPKAPK-2 activation functions as PDK2 for AKT in human neutrophils. J. Biol. Chem., Vol. 276, Issue 5, 3517-3523, 2001 .
Thakkar, R.R., O.-L. Wang, M. Zrouga, W. Stillwell, A. Haq, R. Kissling, W.M. Pierce, Jr., N.B. Smith, F.N. Miller and W.D. Ehringer. Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function. Biochim Biophys Acta Apr 6;1474(2):183-195, 2000 .
Feltzer, R., R.D. Gray, W.M. Pierce and W.L. Dean. Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa: Interaction of Native and N-terminally Truncated Inhibitor Proteins with Pseudomonas Metalloproteinases. J. Biol. Chem., 275(28), 21002-21009, July 14, 2000.
Li J, T-Y Yen, ML Allende, RK Joshi, J Cai, WM Pierce, Jr., E Jaskiewicz, DS Darling, BA Macher and WW Young, Jr. Disulfide bonds of GM2 synthase homodimers: Antiparallel orientation of the catalytic domains. J Biol Chem 2000 Dec 29;275(52):41476-86 .
William M. Pierce Jr., K. Grant Taylor, Leonard C. Waite, Sujan Singh, Jason R. Neale, Xiaoping Tang, and Ned B. Smith. Bone-targeted Estrogens: Anabolic Bone Effects of an Ether-linked 17-O-Estradiol Derivative. J. Bone Mineral Res., 2000
K. Grant Taylor, Jason R. Neale, Sujan Singh, Xiaoping Tang, Peter C. Kulakosky, Valentyn V. Tyulmenkov, Leonard C. Waite, Carolyn M. Klinge, and William M. Pierce, Jr. BONE SELECTIVE ESTROGENS: Estrogen Receptor Alpha Selectivity is a Predictor of in vivo Efficacy. Presented at the annual meeting of the American Chemical Society - Southeast / Southwest Regions. December 2000.
E. Gozal, J.B. Klein, W.M. Pierce, J.A. Scherzer. J.Cai, L.R. Sachleben and D. Gozal. Proteomic analysius of CA1 and CA3 regions of the hippocampus following 6 hours of intermittent hypoxia. Presented at the annual meeting of the Society for Neurosciences.
Klein, J.B., J.J. Williams, J.A. Scherzer, J. Cai, W.M. Pierce and J.M. Arthur. Development of a protein expression database and comparison of rat renal cortical and medullary protein expression using high-throughput proteomic analysis.. J. Am. Soc. Nephrol. 11:409A, 2000.
Arthur, J.M., J.J. Williams, J.A. Scherzer, J. Cai, W.M. Pierce and J.B. Klein. Proteomic identification of proteins involved in magnesium reabsorption in the kidney. J. Am. Soc. Nephrol. 11:557A, 2000.
Song, W., W.M. Pierce, Jr., Y. Saeki, R.A. Prough and R.N. Redinger. Endogenous 7-Oxocholesterol is an Enzymatic Product: Characterization of 7a -Hydroxycholesterol Dehydrogenase Activity of Hamster Liver Microsomes. Arch. Biochem. Biophys., 328: 272-282, 1996 .
Pierce, W.M., Jr., M. Sharir, K.J. Waite, D. Chen and K.K. Kaysinger. Topically active ocular carbonic anhydrase inhibitors: Novel (biscarbonyl)amidothiadiazoles sulfonamides as ocular hypotensive agents. Proc. Soc. Exp. Biol. Med., 203: 360-365, 1993.
Fish, R.H., K.J. Oberhausen, S. Chen, J.F. Richardson, W.M. Pierce, Jr. and R.M. Buchanan. Biomimetic Oxidation Studies. 7. Alkane functionalization with a MMO structural model [Fe2O(OAc)(tris((1-methylylimidazol-2-yl)methyl)amine) 2]3+, in the presence of t-butylhydroperoxide and oxygen gas. Catalysis Letters 18: 357-265, 1993.
King, K.L., N.A. Delamere, S.C. Csukas and W.M. Pierce, Jr.. Metabolism of arachidonic acid by isolated rabbit ciliary epithelium. Exp. Eye Res. 55: 235-241, 1992.
Song, W., W.M. Pierce Jr., R.A. Prough, and R.N. Redinger. Characteristics of cholesterol 7-a -hydroxylase and 7-a -hydroxycholesterol hydroxylase activities of rodent liver. Biochem.Pharmacol. 41: 1439-1447, 1991.
Rodrigues, A.D, D. Fernandez, M.A. Nozarzewski, W.M. Pierce, Jr. and R.A. Prough. Inhibition of hepatic microsomal cytochrome P450-dependent monooxygenase activity by the antioxidant 3-t-butyl-4-hydroxyanisole. Chem. Res. Toxicology 4: 281-289, 1991.
S.D. Gettings, C.B. Brewer, W.M. Pierce Jr., J.A. Peterson, A.D. Rodrigues and R.A. Prough. Enhanced decomposition of oxyferrous cytochrome P450CIA1 (P-450cam) by the chemopreventive agent, 3-tert -butyl-4-hydroxyanisole. Arch. Biochem. Biophys. 276: 500-509, 1990.
T. Yamamoto, W.M. Pierce, Jr., H.E. Hurst, D. Chen and W.J. Waddell. Ethyl carbamate metabolism: in vivo inhibitors and in vitro enzymatic systems. Drug Metabolism and Disposition 18 : 276-280, 1990.
F.W. Benz, D.E. Nerland, W.M. Pierce, Jr. and C. Babiuk. Acute acrylonitrile toxicity: Studies on the mechanism of the antidotal effect of D- and L-cysteine and their N-acetyl derivatives. Toxicol. Appl. Pharmacol. 102: 142-150, 1990.
D.E. Nerland and W.M. Pierce, Jr. Identification of N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine as a urinary metabolite of benzene, phenol, and hydroquinone. Drug Metabolism and Disposition 18: 958-961, 1990.
K.J. Oberhausen, J.F. Richardson, W.M. Pierce, Jr. and R.M. Buchanan. Synthesis, structure and properties of a N3 tridentate bis-imidazolyl ligand with copper(II). Polyhedron 8: 659-668, 1989.
M.S. Mashuta, W.M. Pierce, Jr. and R.M. Buchanan. Binuclear Schiff base macrocycles. Inorg. Chim. Acta 158: 227-237, 1989.
J.S. Hurst, C.A. Paterson, P. Bhattacherjee and W.M. Pierce, Jr. Effects of ebselen on arachadonate metabolism by ocular and non-ocular tissues. Biochem. Pharmacol. 38: 3357-3363, 1989.
L.A. Carr, P.P. Rowell and W.M. Pierce, Jr. Effects of subchronic nicotine administration on central dopaminergic mechanisms in the rat. Neurochemical Research 14: 511-515, 1989.
W.M. Pierce, Jr., A.O. Clark and H.E. Hurst. Determination of ethyl carbamate by gas chromatography with flame ionization or mass spectrometric detection. J. Official Analytical Chemists. 71: 781-784, 1988.
W.M. Pierce, Jr. and D.E. Nerland. Qualitative and quantitative analyses of phenol, phenylglucuronide and phenylsulfate in urine and plasma by gas chromatography/mass spectrometry. J. Anal Toxicology, 12: 344-347, 1988.
M.S. Mashuta, T.N. Doman, W.M. Pierce, Jr. and R.M. Buchanan. Synthesis and characterization of a new binucleating Schiff base macrocycle and its nickel(II) and copper(II) complexes. Inorgan. Chim. Acta 145: 21-28, 1988.
W.J. Waddell, C. Marlowe and W.M. Pierce, Jr. Inhibition of the localization of urethane in mouse tissues by ethanol. Food and Chemical Toxicology 25: 527-531, 1987.
R.D. Gray, W.M. Pierce, Jr., J.W. Harrod, Jr., and J.M. Rademacher. Inhibition of thermolysin by bifunctional N-carboxyalkyl dipeptides. Arch. Biochem. Biophys. 256: 692-698, 1987.
T.I. Senler, W.L. Dean, W.M. Pierce, Jr. and J.L. Witliff. Procedures for measuring Cytochrome P-450-dependent hydroxylation activity in reproductive tissues. Anal. Biochem. 144: 152-158, 1985.
W.M. Pierce, Jr., J.J. Schlager, R.J. Madden and H.E. Hurst. A simple, rapid synthesis of caffeine-1,7-13C. J. Labelled Compounds and Radiopharmaceuticals 21: 187-192, 1984.
We use functional proteomicsstudies to see if genes are doing their jobs
Often, regulatory biology studies rely on determination of gene structure. However, a lot of things happen after transcription and translation, and genomic analysis tells us little about these. A more complete picture is provided by analyzing the “proteome” that is the entire cellular complement of proteins.
At the University of Louisville, there is a Center
for Genetics and Molecular Medicine Some of
its members use these approaches to study interactions of drugs, chemical
environmental pollutants and toxins with living systems.
This section is a narrative description of some of the common techniques of proteomics analysis and some complementary methods.
1. Starting Material – Cell/Tissue lysate or extract Of course the source of the starting material is study dependent. The manner of preparation, however may be critical, and centrifugal methods of cell fractionation and solubilization will have a substantial impact on the success of subsequent analyses.
2. Use of Affinity techniques This is a set of complementary techniques that will typically proceed in parallel to proteomics analyses, including receptor binding, immunoassay, etc. Data obtained from such studies helps guide proteomics data analysis. Here is an example of a scheme used for phosphopeptide analysis.
3. Physicochemical Separation techniques Mass spectrometry is far less useful for the study of mixtures than it is for the study of purified compounds. Thus, it is routinely desirable to “front-end” MS analyses with separation, typically using either chromatography or electrophoesis. In addition to purification, of course, these techniques provide independent measures of one or more physical properties of the analyte.
4. Chromatography Chromatographic separations may be performed using classical techniques (ion exchange, steric exclusion, affinity, etc.) or may be performed using HPLC. The effluent from the HPLC can be introduced directly into the mass spectrometer in many cases, or in parallel with passage through another detection device UV-visible photodiode array, fluorometer, radioactivity detector, electrochemical) which yields other useful data.
5. Electrophoresis For many users, the word proteomics is synonymous with high resolution 2-D electrophoresis with advanced bioinformatics analysis. Following 1-D or 2-D SDS-PAGE, gels are stained with Coomassie Blue, silver stain or Sypro Ruby Red. The results of these analyses are highly purified proteins along with estimates of molecular weight, (MW), isoelectric point (pI) and possible tentative identification. When used along with companion techniques (e.g. 32P-phosphorylation, PVDF transfer and immunoblot, etc.) additional function-related information may be available. All of these measures are used for initial passage through advanced bioinformatics systems for possible ID, followed by robot assisted sample preparation for mass spectrometry (MS) analysis.
6. Structural Analysis Using Mass Spectrometry – Although mass spectrometry is applicable, in principle, to any biomolecule, most of this work will focus on proteins. Following separation of proteins and peptides, mass spectral analyses will be performed for structural elucidation. The 1st goal is identification of known proteins separated by earlier techniques. The 2nd goal is identification of novel proteins. The 3rd goal is identification of sites and types of post-translational modifications (PTM). In this context, PTMs include normal endogenous regulatory changes (e.g. phosphorylation, glycosylation) as well as markers of xenobiotic interactions (e.g. adduct formation or oxidized proteins). Two fundamentally different and complementary mass spectrometric techniques will be employed: (1) matrix-assisted laser desorption ionization followed by time-of-flight (MALDI-TOF) mass analysis and (2) electrospray ionization mass spectrometry (ESI-MS).
Mass
Spectrometry – Analysis Strategy The techniques to be used
will provide data that is complementary to data from chromatrographic and
electrophoretic studies, affinity techniques and classical analyses such
as Edman sequencing. The strategy to be used is a five part sequential
strategy as depicted in the flowchart below. The five components
of this strategy are:
(1) Determine Molecular Weight of the Intact Protein.
Using ESI+-MS an ion envelope for the holoprotein will be obtained, and
an estimate of molecular weight obtained using the Micromass deconvolution
module MaxEnt. In a companion study, MALDI-TOF (linear) data
will be collected on the holoprotein. The two techniques are
complementary in strengths and weaknesses, ESI-MS requires more protein
(pmol) for analysis, has a nominal molecular weight range of up to 100,000
Da and is a higher precision method (these measures typically provide molecular
weight estimates of +/- 2 Da for a 20,000 Da protein.) Linear MALDI-TOF
is much faster, can provide data from femtomol of material and has a nominal
range up to 1,000,000 Da, albeit with less precision.
(2) Determine masses for peptides derived from proteins
of interest. Using the tryptic hydrolysates
described above, MALDI-TOF data will be obtained. Either a Genomic
Solutions or Micromass robot will employ the “dried-drop” and “thin-film
(nitrocellulose)” methods of sample preparation
for MALDI targets using sinapinic acid and alpha-cyanohydroxycinnamic
acid as matrices. Data will be collected in an automated fashion
using a raster array of laser (337 nm) pulses in 10 clusters of 5 averaged
pulses for each sample. MALDI-TOF (reflector) mode will collect
data for m/z = 500-4000, and these data patterns will be compared in an
automated fashion to the Swiss-Prot and EMBL data bases.
(3) Determine partial sequences using PSD experiments.
For proteins that cannot be identified by the pattern matching techniques
described above, MALDI-PSD (post-source-decay) experiments will be conducted.
Often this technique allows us to obtain sequence tags of 3-5 residues.
These sequence tags can then be used to fortify a database search or to
serve as standalone search criteria.
(4) Determine further sequence using ESI-MS-MS.
When operated in the tandem mode (this is a triple quadrupole mass spectrometer)
further de novo sequencing can be performed. This will be performed
for proteins that survive as unknowns to this point.
(5) Structural analysis of post-translational modifications.
Pattern-matching strategies, 2-D PAGE analysis and affinity techniques
described above will highlight the presence of post-translational modifications
of certain peptides. MALDI-TOF PSD and ESI-MS-MS experiments
that focus on these peptides of interest will allow determination of the
MW and charge of the modification, and thus often will allow identification
of the site and type of modification.
Here is a flow chart that depicts a sample analysis strategy
