8/13/2015

1

Mass Spectrometry Workshop

Árpád Somogyi

Associate DirectorCCIC, Mass Spectrometry and Proteomics Laboratory

OSUAugust 17, 2015

8/13/2015

2

Time Monday, 8/17 Tuesday, 8/18

9-10:20amIntroduction to Mass SpectrometryIonization methods (Arpad)

Mass Analyzers (Arpad)

FT-ICR instrumentation and techniques (Mike Freitas)

10:35-12:00pm

GC-MS analysis, examples and EI spectra interpretation (Jeremy)

Ion Activation (Vicki Wysocki)

12-1pm Lunch Break Lunch Break

1-2:30pm Lab visit (Group 1) Lab visit (Group 2)

2:40-4pmHPLC-MS/MS and metabolomics, Data processing programs (Yu Cao)

Small molecule (ESI) spectrum interpretation (Arpad)

Open Discussion/Users Presentations

Workshop schedule

Goal:

attentive class, active learning

Lecture only:

boring, limited active learning

8/13/2015

3

Please interrupt!!

(With questions and comments)

- not with ringing cell phones

Some interactive “learning checks” to keep you engaged will be presented!

What is Mass Spectrometry?

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

– wide variety of compounds (MW: 1-106

daltons) by using

– a small amount of sample (μg-ng, picomol, femtomol)

– easily coupled with separation techniques (GC, HPLC)

Ideal for analysis of complex mixtures

8/13/2015

4

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#).

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)Fast atom bombardment (FAB)

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)

8/13/2015

5

MS:

IonizationIonization AnalysisAnalysis

MS/MS:

Ionization

MS/MS:

Ionization AnalysisAnalysisSelectionSelection Activation

Collide withtarget to producefragments

Activation

Collide with

fragments

Difference between single stage MSand tandem MS/MS

What is Tandem Mass Spectrometry? (MS/MS)

Activate

GasSurfacePhotonsElectrons

Heat

MS1 MS2

8/13/2015

6

Why Sample Preparation ?

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

biofluids, urine, blood. tissues)

Extraction/purification/cleanup

Analysis

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 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 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 Perform ultra sensitive multielement inorganic analyses

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

8/13/2015

7

Mass Spec in Space Science and Astrobiology

The Cassini-Huygens mission is a great success!“Titan is an Organic Paradise” Why?

Origin of Color – Origin of Life????Pre-biotic Earth: pre-biotic chemistry?

The Role of Tholin?

95% N2:5% CH4

with adjustableflow rate

Vacuum pump

Glass tube to collect tholins at 195 K

VAC

Ultrahigh vacuum (UHV) plasma generator to synthesize “tholins” (CxHyNz) in oxygen free environment

8/13/2015

8

FT-ICR of Complex Organic Mixtures (UHVT_001)

99.06649

114.07742

126.07743

141.08831

156.09921

168.09922

181.09448

195.11009

207.11004

222.12071

235.11570

249.15641

259.11514 277.13673

288.14130 301.13655312.14132 341.30364

UHVT_001_ESI_pos_000001.d: +MS

96.88399

114.07766

127.09793

141.08834

155.10399

169.09449

183.11014

195.11020

207.11015

222.12083

234.12064

248.13624

260.13632273.13169

287.14753

300.14296

327.15441 341.17031

UHVT_001_LDI_pos_0_H24_000002.d: +MS0.0

0.5

1.0

1.5

2.0

2.5

8x10Intens.

0.0

0.5

1.0

1.5

2.0

8x10

100 150 200 250 300 350 m/z

Positive Ions: ESI

Positive Ions: LDI

108.03146

119.03627

141.02065

160.03772

185.05814

198.05341

209.03302

224.06911

239.08001 251.08001

265.14795

283.26429

293.17921

306.09696

321.21053 347.12280

UHVT_001_ESI_neg_000001.d: -MS

141.02059

165.02070

192.03152

209.03285

224.06904

233.03311 251.05489

264.07533275.05486 306.09696 318.09726328.10639

345.10840

UHVT_001_LDI_neg_0_I24_000003.d: -MS0.00

0.25

0.50

0.75

1.00

1.25

1.50

9x10Intens.

0

1

2

3

4

9x10

100 150 200 250 300 350 m/z

Negative Ions: ESI

Negative Ions: LDI

Not the same protonated/deprotonated neutral species in +/- modes

8/13/2015

9

Modified van Krevelen diagram for LDI ions

8/13/2015

10

Example of ultrahigh resolution in an FTICR

J. Throck Watson “Introduction to Mass Spectrometry” p. 103

8/13/2015

11

N2+.

C2H5+

HN2+

N2/CH4 (5%)2s,5x10-7 torr15s EI ionization with 40eV

HCNH+CH2=CH2

+.

Mass Spectrometry Imaging

8/13/2015

12

Selection of [M+2H]+2 at m/z 246.1

100

80

60

40

20

0Rel

ativ

e In

ten

isty

76543210Drift Time (ms)

600

500

400

300

200

100

m/

z

SDGRGGRGDS

8/13/2015

13

Transfer CID of [M+2H]+2 at m/z 246.1

100

50

0

Rel

ativ

e A

bu

nd

ance

500400300200100m/z

100

50

0

3.0

2.5

2.0

Dri

ft T

ime

(ms)

dt = 2.49-2.68 ms

dt = 2.68-2.83 ms

GRGDS

SDGRG

b4

b3b2

y1

R

R

Si

Si

y4y3

Fragmentation of Big Protein Complexes

8/13/2015

14

Ionization Methods

Neutral species → Charged species

• Removal/addition of electron(s)– M + e- → (M+.)* + 2e-

• electron ionization

• Removal/addition of proton(s)– M + (Matrix)-H → MH+ + (Matrix)-

• chemical ionization (CI)• atmospheric pressure CI (APCI)• fast atom bombardment (FAB)• electrospray ionization (ESI)• matrix assisted laser desorption/ionization (MALDI)• desorption electrospray ionization (DESI)• direct analysis in real time (DART)

Still widely used in

Forensic EnvironmentalDrug Metabolism, etc.

Electron Impact (EI) Ionization

8/13/2015

15

8/13/2015

16

The Nitrogen Rule

• Compounds* that contain even number of N atoms have even number of nominalmolecular weight

• Compounds* that contain odd number of N atoms have odd number of nominalmolecular weight

But what about singly protonated moleculesand accurate molecular weights??

* Common organic compounds

Ion Stabilities

• Even electron ions are more stable than odd electron (radical) ions

How about protonated molecules: even electron or not?

And how about ions formed by electron impact (EI) ionization?

8/13/2015

17

• Filament: tungsten or rhenium

• Current: 3-5 A

• Ufil: a few Vs (1-3 V)

• Tfil: ca. 2000-2300 K (ca. 150 0C in the ion source)

• Source Block

• pressure: ca. 10-5 torrs (mean free path ca. 100 m)

• repeller: 1 V, m/z 100 u, distance 0.1 cm

– tres = 1.4x10-6 s

8/13/2015

18

Chemical Ionization

• Ion-molecule reaction(s) between a reagent gas and the sample at a relatively high pressure

• Most common reagent gases– methane, isobutane, ammonia

• Mechanisms– CH4 + e- → CH4

+., CH3+, CH2

+., …– CH4

+ + CH4 → CH5+ + CH3

– CH3+ + CH4 → C2H5

+ + H2

– CH5+ + M → [M+H]+ + CH4

– C2H5+ + M → [M-H]+ + C2H6

– C2H5+ + M → [M+ C2H5]+

8/13/2015

19

Protonation is one type of ionization

M + AH+ → MH+ + A

CH3CH2NH2 + (NH3)nNH4+ → CH3CH2NH3+ + (n-1) NH3

The extent of fragmentation depends on the exothermicity of the reaction

Proton affinity (PA):

M + H+ → MH+ - ΔHr = PA

8/13/2015

20

Proton affinity (PA):

M + H+ → MH+ - ΔHr = PA

PAs of common CI reagents (kcal/mol)methane (131) < water (173) < methanol (185) < CH2=C(CH3)2 (197) < ammonia (205)

If the analyte has a much higher PA than that of the unprotonated reagent, the protonation of the analyte will be (very) energetic (fragment rich CI spectra, “semi-CI”)

8/13/2015

21

Sources still being developedDESI- desorption ESI

(sample not in solution)

DART

8/13/2015

22

DARTDirect Analysis in Real Time

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

Can we teach elephants to fly?John Fenn

8/13/2015

23

Yes, of course!!!Nobel Price in Chemistry, 2002

John B. Fenn Koichi Tanaka

Electrospray IonizationESI

Soft Laser DesorptionSLD

Matrix Assited LaserDesorption/IonizationMALDI

8/13/2015

24

Matrix-Assisted Laser Desorption\ Ionization

MATRIX - A small acidic organic molecule (matrix) is mixed with a low concentration of analyte in a common solvent and allowed to co-crystallize on a sample plate to form a “solid solution” by which the analytemolecules are isolated from each other.

ASSISTED – matrix “assists” the desorption and ionization of the analyte(s)

LASER – Typically a Nitrogen laser (351 nm) or Yag/Nd laser (334 nm)

DESORPTION – Energy from the laser desorbs the matrix into the gas-phase and “carries” the analyte with it.

IONIZATON - Detect [M+H]+ by transferring a proton from the matrix to the analyte

Choose a matrix based on the molecular weight, solubility and chemical structure of the analyte.

Excellent for intact molecular weight determination for both polar and non-polar molecules with mass > 500 amu.

Cation (H+, Na+ etc)

Matrix

Analyte

Pulsed Laser

Sample Plate

Desorbed plume of matrix and analyte ions

Extraction Grid to TOF

MALDI MatricesMatrix Abbrev Sample Type

2,5-dihydroxybenzoic acid DHB Peptides < 5,000 polymers, dedrimers

Good universal matrix“cold matrix”

50% ACN in 0.1% TFA,THF, 2:1

chloroform:MeOH

3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid)

SA Peptides and Proteins > 10,000

“hot matrix”

50-70% ACN in 0.1% TFA

-cyano-4-hydroxycinnamic acid HCCA Excellent for peptides, digestion products

and proteins

50% ACN in 0.1% TFA

Dithranol Non-polar polymers THF, Methylene chloride

Indoleacrylic acid IAA Non-polar polymers THF, methylene chloride

3-hydroxypicolinic acid HPA DNA and negative ion samples

See me for more specific procedure

Trihydroxyacetophenone THAP DNA and negative ion samples

See me for more specific procedure

Nor-harmane Universal 50% ACN, THF, chloroform

8/13/2015

25

The MALDI Plate with Different Samples in Different Matrices

8/13/2015

26

2 GHz time digital converter1000 Hz laser (acquisition of 1000 spectra in 1 second

10000 20000 30000 40000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

[2M+H]+

14318.68

[M+2H]2+

7157.18

[M+H]+

14318.68

Inte

nsity

m/z

MALDI-TOF spectrum of a pure protein (linear mode)

8/13/2015

27

5000 10000 15000 20000 25000 30000 35000 40000 m/z

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

a.i.

/export/home/tof/data/mslab/091705/pro_6/1Lin/pdata/1 unknown Thu Oct 6 14:27:19 2005

Exercise 2This is a MALDI-TOF spectrum of two proteins. Identify the proteins by

molecular weights and assign the labeled ions

5392.3

9757.4

6267.7

9080.3

4373.8

4880.1

3133.7

7288.2

3645.17884.4

3941.6

4620.1

8380.3 10314.3

9508.3

8833.8

0

2000

4000

6000

8000

Inte

ns.

[a.u

.]

3000 4000 5000 6000 7000 8000 9000 10000 11000m/z

MALDI-TOF spectrum (protein profile) of a bacteria

8/13/2015

28

Molecular weight distribution defines polydispersity of polymers

Polydispersity (PD) = Mw/Mn

Polystyrenewith Ag+

Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, 275-281.

829.987

991.986

1143.205

1313.231

1433.246

1553.257

1723.288

1123.212

2013.333

2303.4052593.467

2933.604

* NB_89_M4\0_M16\1\1SRef, "Baseline subt."

0.0

0.5

1.0

1.5

2.0

5x10

Inte

ns.

[a.u

.]

1000 1500 2000 2500 3000 3500 4000 4500m/z

Trancated Vectra Polymer (Somogyi et. al., unpublished)

PD > 1.05

PD≈1.01

Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, 275-281.

MS is an absolute method for MW determination(but remember possible degradation!)

8/13/2015

29

Problems with polymer analysis• Sample preparation

– Several polymers are not well soluble in conventional solvents

– Solventless technique– Cationizing with Na+, K+, and Ag+ by spiking– Synthesis of tailor (home) made matrices

• Degradation during ionization (especially with MALDI)– Photodissociation in MALDI (MALDI/ESI comparison

desirable)

• End group analysis reliable but better to have high resolution/accurate mass (FT-ICR)

Some Examples for Tailor-Made Matrices

NN

FF

F F

HOCOOH

M-1

NN

FF

F F

FF

FF

F FM-3

FF

F F

HOCOOCH3

M-2

NN

FF

F F

HOOH

FF

F FM-4

FF

F F

HOOH

FF

F F

M-5

FF

F F

FF

FF

F F

M-6

FF

F F

FCOOCH3

M-7

NN

FF

F F

H3COOCH3

FF

F FM-9

NN

FF

F F

HOCOOCH3

M-8

Somogyi, et al., Macromolecules, 2007, 40, 5311-5321.

8/13/2015

30

http://www.polymerprocessing.com/polymers/alpha.html

Poly(ethylene terephtalate) (PET)C10H8O4 (192.042259)

Polystyrene (PS)C8H8(192.042259)

Poly(methyl methacrylate) (PET)C5H8O2 (100.052430)

Poly(vinyl alcohol) (PVA)

C2H4O (44.026215)

- CH2-CH2-O-Poly(ethylene glycol) (PEG)

Repeating unit masses of polymers

0

1000

2000

3000

4000

Inte

ns.

[a.u

.]

500 1000 1500 2000 2500 3000 3500m/z

0

500

1000

1500

2000

Inte

ns.

[a.u

.]

1675 1700 1725 1750 1775 1800 1825 1850 1875m/z

Synthetic Polymer Analysis by MS (MALDI-TOF)

1684.205

1728.2311772.260

1816.2921660.310

4444

4444

8/13/2015

31

CH3OH exact mass: 32.0262 u

Electrospray Ionization (ESI)

++ + + ++

+++

+ + + +++

+++

+

Evaporation ++++

+++++

+

+

+

Columbic Explosion

Critical RadiusCharge Repulsion > Surface Tension

++

++ +++

+

+++

Fused silica

Metal Capillary3-5 kV

An electric field on the capillary produces a spray of fine charged droplets The presence of a drying gas (Nitrogen) and heat evaporates off the solvent leaving a distribution of multiply charged de-solvated ions. Soft ionization In-line compatible with HPLC

8/13/2015

32

Z1 = j(p2 – Ma)p1 – p2

Z1 = 1(1030.8 – 1)(1124.6 - 1030.8)

Z1 = 10.978 round up to 11Therefore, p1 has 11 charges (protons)

Pick as p1

Pick as p2

Calculate intact molecular weight Mr = p1 z1 – Maz1Mr = (1124.6 * 11) – (1 * 11)Mr = 12359.6 (Actual for Cytochrome C is 12360.1

For any peak j charges away

8/13/2015

33

ESI-MS of Myoglobin

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

A15A16

A17

A18

A19

A20

A21

A14A13

A12

A11

A10

10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000mass74

100

%

A

m/z deconvolutesto16952

ESI vs MALDI

Choose MALDI when: Peptides or protein with a MW < 5000 Da (MALDI can

detect MW > 100KDa, but not very accurately)

Complex mixtures (more than 5 compounds)

Very little material with higher salt/buffer concentration

Choose ESI when:MW > 5000 Da (proteins)

Want better mass assignment

Want good MS/MS data

8/13/2015

34

Dual ionization mode on the apex-Qe

Apollo™ II with MALDI Option