Mass Spectrometry Infrastructure
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1 Mass Spectrometry Infrastructure Todd Williams, Ph.D. Director KU Mass Spectrometry and Analytical Proteomics Laboratory Mass Spectrometry Lab B025 Malott Hall
2 Mission The Mass Spectrometry and analytical Proteomics Laboratory (MSL/APL) is a research resource with a primary mission to provide collaboration and service in chemical analysis by mass spectrometry and to educate investigators on the uses of mass spectrometry as a research tool. Mass Spectrometry Lab
3 Lab utilization in three major categories Synthetic confirm formula and support structure of something you made Target identification and quantitation Is my molecule of interest in Bio Fluid X Omics and complex projects Lipidomics, Proteomics : if we measure everything an answer should emerge? MSL/APL tradition is to offer practical state of the art separation techniques as well as tandem mass spectrometry. Engage investigators to use post acquisition software and interrogate their own data. Mass Spectrometry Lab
4 1 st of many questions: What Ionization methods? EI (Electron Ionization) hard lots of energy for fragmentation with structure information But molecular weight not always represented no ion at m/z 180 Thus consider CI, FAB, MALDI, ESI are soft er Source GC region Magnet ESA Magnetic Sector Instrument fw=180 8 feet A dinosaur.but turtles and crocs still roam the earth! Installed in 1985 Mass Spectrometry Lab B025 Malott Hall
5 2 nd choice Instrumentation consideration: What might a user care about after ionization? 10,000 resolution Resolution Mass accuracy 5000 resolution Quadrupole resolution Mass Spectrometry Lab B025 Malott Hall Mass Spectrometry Lab
6 Ubiquitin on LTQ FT our ultimate resolution instrument F # RT: AV: 3 NL: 2.08E5 T: FTMS + p ESI Full ms [ ] M9H 9+ = u apart also sub ppm accuracy w/ effort Relative Abundance F #14-16 RT: AV: 3 NL: 4.84E5 T: FTMS + p ESI Full ms [ ] Relative Abundance z= z= z= m/ z z=6 FWHH=.006u z=5 z= Res=m/ m=952/.006= 159, m/z Mass Spectrometry Mass Spectrometry Lab B025 Malott Lab Hall
7 Synthetic Support I have a mass. now what? Nominal Mass assignment (+ 1 u) confirms Hydrogen count in CHNOX assignment theoretical Formula confirmation with exact mass measured For C H 3-74 O 0-4 N 0-4 Mass 118 Mass 500 Mass next formula 34 ppm 5 compounds within 5 ppm 626 compounds within 5 ppm Note depending on mass the exact mass is necessary but not sufficient to prove a formula Software searches formula Mass Spectrometry Lab B025 Malott Hall
8 Synthetic Support Let s do an experiment MH + Chemists Ideal MS spectrum What users want MNa + Real Electrospray spectrum What they get 2M(H,K,Na) + MSL can help. What is that high mass stuff? Mass Spectrometry Mass Spectrometry Lab B025 Malott Lab Hall
9 CaMox Synthetic support Extends to biopolymer modification Calmodulin (CaM) oxidation and nitration Tryptic digest VFDKDGNGYISAAELR (T9) CaM(WT)-ox T9 3+ (T9+NO 2 ) 3+ CaM(L9)-ox T9 3+ +NO 2 (T9+NO 2 ) 3+ H. S. Smallwood et al," Selective Nitration of Tyr 99 in Calmodulin as a Marker of Cellular Conditions of Oxidative Stress" Chem. Res. Toxicol. 2003, 16, Mass Mass Spectrometry Spectrometry Lab Lab B025 B025 Malott Malott Hall Hall
10 Many samples look better with a LC separation coupled to MS Whole protein separation on C 4 reverse phase. Oxidized calmodulin. Capillary HPLC Qtof Mass spectrometer N. A. Galeva et al Rapid method for quantifying the extent of methionine oxidation in intact Calmodulin J. Am. Soc. Mass Spectrom. 2005, 16, Mass Spectrometry Lab B025 Malott Hall
11 Red Blood Cells n= Ion pair UPLC Chromatography and MS on a triple quadrupole n Target ID and quantitation QQQ instrument UPLC >8000 psi small particles Narrow peaks, short total resolution time Poly glutamate metabolites of methotrexate in red blood cells L. van Haandel, et al Rapid Commun. Mass Spectrom. 2009; 23: Mass Spectrometry Lab B025 Malott Hall
12 Adventures in Sampling Chemistry student Prescription drugs in waste water Metoprolol m/z Propranolol m/z Atenolol Albuterol (X 10) m/z m/z Fluoxetine m/z K.D. Bratton, A.S. Lillquist, T.D. 0Williams and 2 C.E. Lunte 4, " The Determination 6 and 8 Quantification 10 of Human 12 Pharmaceutical 14 in 16 Aquoeus Environmental Samples", Chapter 12 in "Mass Spectrometry, LC/MS/MS and TOF/MS :Analysis of Emerging Contaminants" ACS Symposum Volume 850, Imma Ferrer and E.M. Thurman, Eds (2003), Oxford University Press and ACS. p Mass Spectrometry Lab
13 OMICS Primary Data in Informatics Measure all modified lipids and proteins Lipids& Proteins: hepatocytes treated with a mixture of TASO and [ 13 C 2 D 3 ]-TASO isotopic label helps! MS/MS spectra assigned to acetyl adducts of AAYQVAALPK(+42/47)GSR (MW ) peptide lipid protein [ 13 C 2 D 3 ] = +5u Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites. Sarma, D. et al Chem Res Toxicol : Mass Mass Spectrometry Lab Lab B025 B025 Malott Hall Hall
14 How to Submit Samples reqform2.doc B025 or Data stations for work-up by clients Samples for SBC Room 1086 Mass Spectrometry
15 How to get Good Data! Three keys sample prep sample prep sample prep Feed back from Staff What do you want to learn from the sample? Get help in assessment of data.good looking spectra..or junk? Controls Parallel samples to test manipulations and sensitivity. Practice Samples Use abundant cheap sample related to project. Spike with reporter molecule for post biology artifacts. Mass Spectrometry Lab
16 Contact Info Todd Williams, Ph.D. Director KU Mass Spectrometry and Analytical Proteomics Lab 3006 Malott Hall Structural Biology Center Nadezhda Galeva, Ph.D. Research Associate MS & APL SBC room Lawrence Seib Research Assistant Robert Drake Research Instrument Operator Malott rooms B Labs Mass Spectrometry Mass Spectrometry Lab B025 Malott Lab Hall
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