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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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

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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

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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