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Mass Spectrometry WorkshopOhio State University

July 28-30, 2014Árpád Somogyi

Associate Director, CCIC Mass Spectrometry and Proteomics

Introduction, Peptide fragmentation/sequencing, Ionization Methods, Mass analyzers, Derivatization, Small molecule analysis, Imaging, Ion mobility, Synthetic Polymers, Metabolomics, Clinical applications

Liwen Zhang

Project Manager for Proteomics, Research Scientist

Proteomics I-III

Cindy James

Senior Research Associate

Sample Preparation, Protein Purification, Protein Digest

http://www.ccic.ohio-state.edu/MSP

Mass Spec & Proteomics•About CCIC MS&P•Rates & Forms•Our Staff•Protein Expression and Purification•Proteomics•Services•Instruments•Bioinformatics•Driving and Parking Info•Acknowledging the MS&P Facility•Useful Links•Scheduling/FOM

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MSP = Mass Spectrometry and Proteomics

Goal:

attentive class, active learning

Lecture only:

boring, limited active learning

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Please interrupt!!

(With questions and comments)

- not with ringing cell phones

We’ve planned some interactive “learning checks” to keep you engaged.

What is Mass Spectrometry?

• Mass spectrometry is a powerful tool in analytical/bioanalytical chemistry that provides– detailed structural information for a

– wide variety of compounds (MW: 1-106 daltons) by using

– a small amount of sample (ug, picomol, femtomol)

– often and easily coupled with separation techniques (GC, HPLC) – ideal for mixture analysis

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What can we provide by mass spec?

• MW determination– nominal– accurate (elemental composition)

• isotope pattern• high resolution

• Fragmentation– fragmentation rules– libraries (“fitting”)– MS/MS (or MSn)

• Thermodynamic parameters– ionization energy (IE)– appearance energy (AE)– heats of formation (∆Hf)– activation enthalpy (∆H#), activation entropy (∆S#)

What do we measure by mass spectrometers?

Mass to charge (m/z) ratios!

Average (chemical) molecular mass

Accurate mass

Isotope Distribution

Is that all????

No!! (You will see why….)

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Want to do MS or MS/MS ?Need a Mass Spectrometer

inlet all ions

Ionizationsource

Massanalyzer

sortedions

Detector

Datasystem

Ionization Source Mass Analyzer Detector Computer

Sample IntroductionDirect probe/infusion

GCHPLC

Sample Preparation

Vacuum System

Electron impact (EI)Chemical ionization (CI)Atmospheric pressure (API)Electrospray (ESI)Matrix assisted laser Desorption/ionization (MALDI)Surface enhanced LDI (SELDI)Desorption Electrospray (DESI)Direct Analysis Real Time (DART)

Electrostatic (ESA)Magnet (B)Time-of-flight (TOF)Quadrupole (Q)Ion Traps (2D & 3D IT)Ion-CyclotronResonance (ICR)Orbitrap (OT)

Rough, Turbomolecular, andCryo pumps

Electron MultiplierPhotomultiplierFaraday capArray DetectorsMultichanel plate (MCP)

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Why Sample Preparation ?

Analytes of interest found invariety of matrices (e.g.,

biofluids, urine, blood. tissues)

Extraction/purification/cleanup

Analysis

If not……

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Ion Currentover 60 min

MS

MS/MS

26.47

571.29

HPLC chromatogram

MS:

IonizationIonization AnalysisAnalysis

MS/MS:

Ionization

MS/MS:

Ionization AnalysisAnalysisSelectionSelection Activation

Collide withtarget to producefragments

Activation

Collide with

fragments

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MS/MS means selection & fragmention

ActivateMS1 MS2

Simulation of Two-Step Processhttp://monte.chem.ttu.edu/index.html/animations/html/ani-digli-5.html

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What is Mass Spec good for? Detect and identify the use of steroids in athletes Monitor the breath of patients by anesthesiologists during surgery Determine the composition of molecular species found in space - astrobiology Determine whether honey is adulterated with corn syrup Locate oil deposits by measuring petroleum precursors in rock Monitor fermentation processes for the biotechnology industry Detect dioxins in contaminated fish Determine gene damage from environmental causes Establish the elemental composition of semiconductor materials Identify structures of biomolecules, such as carbohydrates, nucleic acids and

steroids Sequence biopolymers such as proteins and oligosaccharides Determine how drugs are used by the body - pharmacokinetics Perform forensic analyses such as conformation and quantitation of drugs of abuse Analyze for environmental pollutants Determine the age and origins of specimens in geochemistry and archaeology Identify and quantitate compounds of complex organic mixtures – natural products Perform ultra sensitive multielement inorganic analyses

http://www.asms.org/whatisms/p1.html

DART(Direct Analysis in Real Time)

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DARTDirect Analysis in Real Time

Penning Ionization M* + S S+. + M + electron (but also allows MH+, M-H-, etc)

Time Monday, 7/28 Tuesday 7/29 Wed 7/30

9-10:20amIntroduction to MSSample Prep I(protein purification)

Proteomics III(Quantitation)

Small molecule analysis

10:35 -11:50am

Sample Prep II(protein separation,modification)

Ionization methodsInstrumentation I(ion sources, detectors)

ImagingIon mobilityIRMPD, HDX

12-1pmLab visit(Group 1)

Lab visit(Group 2)

Lunch break

1:30-2:35pmPeptide fragmentation(manual sequencing)Proteomics I

DerivatizationSample Prep for small molecules

Synthetic polymers (end group analysis)Astrobiology

2:50-4pmProteomics II(PTMs)

Instrumentation II(mass analyzers)

MetabolomicsBiomarkers,Carbohydrates, DNA

What are we going to talk about?Course Outline

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accurate & precise precise, not accurate

accurate, not precise not accurate, not precise

Mass Spectrometry can be very precise, must be accurate

FTICROrbitrapTOF

QuadrupoleIon Trap

For each of the following applications, choose the most appropriate mass analyzer from the following list. Use each analyzer only once.

orbitrap (OT) quadrupole (Q)time-of-flight (TOF) FTICR

Analyzer Purpose______ synthetic organic chemist wants accurate

(exact) mass of a compound_______ biochemist wants protein molecular weight of

relatively large protein (MW 300,000)_______ EPA (Environmental Protection Agency) wants

confirmation of benzene in extracts from 3000 soil samples

_______ Petroleum chemist wants to confirm the presence of 55 unique compounds at onenominal mass/charge value in a mass spectrum

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Selected Isotope Ratios and 13C Contributions

Selected Isotope Ratios and 13C Contributions

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3 5 7 4 3 5 7 6 3 5 7 8 3 5 8 0 3 5 8 20

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

3 7 4 3 7 6 3 7 8 3 8 0 3 8 20

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

(Ala)5

C15H29O6N5

375.2118 (exact mass)

375.4263 (average mass)

3 5 7 4 3 5 7 6 3 5 7 8 3 5 8 0 3 5 8 20

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

3 7 4 3 7 6 3 7 8 3 8 0 3 8 20

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

1 9 7 6 1 9 7 8 1 9 8 0 1 9 8 2 1 9 8 40

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

1975.9541 (exact mass)

1977.1722 (average mass)

(Ala)25

C90H129O26N25

3569.8663 (exact mass)

3572.0062 (average mass)

(Ala)50 C150H252O51N50

3568 (nominal mass)

(Ala)25 C90H129O26N25

3568 (nominal mass)

3 7 4 3 7 6 3 7 8 3 8 0 3 8 20

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

3 5 7 0 3 5 7 2 3 5 7 4 3 5 7 6 3 5 7 80

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

a

b

374.4183 (average mass)

374.2040 (exact mass)

3570.8741 (exact mass)

3572.9742 (average mass)

0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0

0

1

2

3

4

5

6

7

8

De

via

nc

efr

om

no

min

al

ma

ss

(d

alt

on

)

m / z

c

[Ala5+H]+

[Ala50+H]+

exact mass

average mass

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Resolution

http://www.matrixscience

Resolution

30,000

10,000

3,000

1,000

MALDI-TOF Spectrum of tryptic digest(peptide mass mapping)

m/z500 2500

90

0

x 4.0

Ref

Ref

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Newborn Screening by MS/MS

“Currently, Neo Gen Screening monitors more than 60 chemical and chemical relationships in a single test.”

“Tandem Mass Spectrometry measures these indicators or markers in a typical 2-minute run.”

http://www.neogenscreening.com/index.cfm

Product Ion Scan of Phenylalanine butyl ester shows loss of m/z 102

Ref: Chace, 2001

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Control

Phenylketonuria

Blood sample fromNormal Newborn

Masses of deuterated internalstandards are underlined

Blood sample fromnewborn diagnosedwith phenylketonuria

Ref: Chace, 2001

Neutral Loss Spectra from Newborn Screen

serine

threonine

C 12

H 1

O 16

P 31

16+16+16+31 = 79

phosphorylationadded to serine:79-1+2 = 80

could be lost from serine(as an ion):79 (PO3

-)

could be lost from serine(as a neutral):80 + 18 (H2O)= 98

MS/MS Scan Modes Strategy: Phosphorylationof serine, threonine or tyrosine

tyrosine