Post on 07-Jun-2020
Mass Spectrometry Infrastructure
Todd Williams, Ph.D.Director KU Mass Spectrometry and Analytical
Proteomics Laboratory
Mass Spectrometry LabB025 Malott Hall
msl.ku.edu
Mission
Mass Spectrometry Lab msl.ku.edu
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.
Lab utilization in three major categoriesSynthetic confirm formula and support structure of something you made
Target identification and quantitationIs my molecule of interest in “Bio Fluid X”
“Omics” and complex projectsLipidomics, Proteomics : if we measure everything an answer should emerge ?
Mass Spectrometry Labmsl.ku.edu
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 B025 Malott Hall msl.ku.edu
MagnetESA
GCSource region
8 feet
Magnetic Sector Instrument
A dinosaur….but turtles and crocs still roam the earth!
EI (Electron Ionization) “hard”lots of energy for fragmentationwith structure information
But molecular weight not always represented ……no ion at m/z 180
Thus considerCI, FAB, MALDI, ESI are “soft”er
Installed in 1985
1st of many questions: What Ionization methods?
fw=180
Mass Spectrometry Lab B025 Malott Hall msl.ku.eduMass Spectrometry Lab
msl.ku.edu
Quadrupole resolution
5000 resolution
10,000 resolution
Resolution
Mass accuracy
2nd choice Instrumentation consideration:What might a user care about after ionization?
Ubiquitin on LTQ‐FT our ultimate resolution instrument
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Mass Spectrometry Labmsl.ku.edu
F110902 #14-16 RT: 1.42-1.63 AV: 3 NL: 4.84E5T: FTMS + p ESI Full ms [ 190.00-2000.00]
1000 1100 1200 1300 1400 1500 1600m/z
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Rel
ativ
e A
bund
ance
1224.53z=7
1071.59z=8
1428.29z=6
952.53z=9
1661.07z=0
F110902 # 14-16 RT: 1.42-1.63 AV: 3 NL: 2.08E5T: FTM S + p ESI Full ms [ 190.00-2000.00]
952.2 952.4 952.6 952.8 953.0 953.2m/z
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Rel
ativ
e Ab
unda
nce
952.53
952.64 952.75
952.42
952.86
952.97952.30
953.08
FWHH=.006u
Res=m/m=952/.006=
159, 000
M9H9+=
.11u apart
z=5
952.2 953.2
also sub ppm accuracy w/ effort
Synthetic Support I have a mass…. now what?
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
theoretical
measuredFormula confirmation with exact mass
For C0-100 H 3-74 O0-4 N 0-4Mass 118 next formula 34 ppm
Mass 500 5 compounds within 5 ppm
Mass 750.4 626 compounds within 5 ppm
Note depending on mass the exact mass is necessary but not sufficient to prove a formula
Nominal Mass assignment (+ 1 u) confirmsHydrogen count in CHNOX assignment
Software searches formula
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Mass Spectrometry Labmsl.ku.edu
MH+
2M(H,K,Na)+
MNa+
Chemists’ Ideal MS spectrum
What users want
Real Electrospray spectrumWhat they get
Synthetic Support
MSL can help.What is that high mass stuff?
Let’s do an experiment
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
VFDKDGNGYISAAELR(T9)CaMox
Tryptic digest
T93+CaM(WT)-ox
CaM(L9)-oxT93+
+NO2
H. S. Smallwood et al ," Selective Nitration of Tyr99 in Calmodulin as a Marker of Cellular Conditions of Oxidative Stress" Chem. Res. Toxicol. 2003, 16, 95-102
Synthetic supportExtends to biopolymer modification
Calmodulin (CaM) oxidation and nitration
(T9+NO2) 3+
(T9+NO2) 3+
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Whole protein separation on C4 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, 16,1470-1480
Many samples look better with a LC separation coupled to MS
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
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Ion‐pair UPLC Chromatography and MS on a triple quadrupole
Red Blood Cells
n=
n
L. van Haandel, et al Rapid Commun. Mass Spectrom. 2009; 23: 3693-3702.
UPLC >8000 psi small particlesNarrow peaks, short total resolution time
QQQ instrument
Poly glutamate metabolites of methotrexate in red blood cells
Target ID and quantitation
Mass Spectrometry Lab msl.ku.edu
Prescription drugs in waste water
Adventures in Sampling
Chemistry student
K.D. Bratton, A.S. Lillquist, T.D. Williams and C.E. Lunte , " The Determination and Quantification of Human Pharmaceutical in 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. p188-206
0 2 4 6 8 10 12 14 16
Albuterol (X 10)
240 – 148 m/z
Atenolol
267 – 145 m/z
Metoprolol
268 – 116 m/z Propranolol
260 – 116 m/z
Fluoxetine
310 – 44.3 m/z
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Lipids& Proteins: hepatocytes treated with a mixture of TASO and [13C2D3]-TASO isotopic label helps!
[13C2D3] = +5u
MS/MS spectra assigned to acetyl adducts of AAYQVAALPK(+42/47)GSR (MW 1372.7463) peptide
“OMICS” Primary Data in Informatics
“Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites.” Sarma, D. et al Chem Res Toxicol. 2012 25:1868-77 .
Measure “all modified lipids and proteins”
lipid
protein
Mass Spectrometry msl.ku.edu
http://129.237.137.185/prolims2/index.php/site/login
http://www.msl.ku.edureqform2.doc B025 or 3007
Samples for SBCRoom 1086
How to Submit Samples
8 Data stations for work-up by clients
Mass Spectrometry Lab msl.ku.edu
How to get Good Data!
Feed back from StaffWhat do you want to learn from the sample? Get help in assessment of data ….good looking spectra …..or junk?
Practice SamplesUse abundant “cheap” sample related to project.Spike with reporter molecule for “post biology artifacts”.
ControlsParallel samples to test manipulations and sensitivity.
Three keys sample prep
sample prepsample prep
Contact Info
Mass Spectrometry Lab B025 Malott Hall msl.ku.edu
Mass Spectrometry Lab msl.ku.edu
mslab1@ku.edu785-864-3280Malott rooms B025 3006Labs 3002 3006 1008
Todd Williams, Ph.D.Director KU Mass Spectrometry and Analytical Proteomics Lab3006 Malott Hall 785-864-32231083 Structural Biology Center 785-864-3152twilliams@ku.edu
Nadezhda Galeva, Ph.D.Research Associate MS & APLSBC room 1088 785-864-3691galeva@ku.edu
Lawrence SeibResearch Assistant
Robert DrakeResearch Instrument Operator