Mass Spectrometry on the Cinco de Mayo - Rutgers...

Mass Spectrometry on the Cinco de Mayo

Peter Lobel, Ph.D.

Resident Member, Center for Advanced Biotechnology and Medicine

Professor of Biochemistry and Molecular Biology, Rutgers-RWJMS

Director, Rutgers-RWJMS Biological Mass Spectrometry Facility

Member, Center for Integrative Proteomics Research

[email protected]

Images courtesy of David Fenyö, Nathan Yates, Wilhelm Haas, Michael

McCoss, AB-SCIEX, Thermo

mailto:[email protected]

Outline of today’s talk

• Overview - mass spectrometry in proteomics

• How mass spectrometers work

• Looking at spectra - mass and isotopes

• Quantitative proteomics

• Measures mass/charge (m/z) of intact ions and

fragments

What does a mass spectrometer do?

Ion

Source

Mass

Analyzer Detector

m/z

inte

nsit

y

Structural

information

Quantitative

information

vacuum

Use of mass spectrometry in proteomics research

protein identification

protein processing:

proteolytic

post-translational modifications (100’s)

quaternary structure

complex formation

steady-state levels

turnover rates

structural studies (H/D exchange)

comparison of samples (disease & controls)

Mass spectrometry is an enabling technology*

in the field of proteomics

Peptide Sequencing

Edman degradation Mass spectrometry

Material required ~pmol

(6 x 1011 molecules)

fmol

6 x 108 molecules

Time (data collection) ~10 hours 10 msec

Wikipedia

*An enabling technology is an invention or innovation, that can be applied to drive

radical change in the capabilities of a user or culture. Enabling technologies are

characterized by rapid development of subsequent derivative technologies, often in

diverse fields.

Equipment and/or methodology that, alone or in combination with associated

technologies, provides the means to increase performance and capabilities of the

user, product or process.

http://en.wikipedia.org/wiki/Invention

http://en.wikipedia.org/wiki/Innovation

400 600 800 1000 1200 1400 1600 1800m/z

Re

lative

Ab

undan

ce

MS

1. MS1 (MS) spectra measure

intact peptide ions

500 300 700 900

507.2962 K.RLNIVQDR.F

507.3031 K.ANELLINVK.Y

507.3031 K.IIAIDINNK.K

507.3031 R.VLNLPSVGSK.S

507.3088 R.LNVLSNVVR.K

507.3088 K.SPKSNKKPK.R

507.3213 K.AIILGAQSIK.C

...

Predicted tryptic peptides in database

507.303

observed m/z, z=2 The precursor mass is not

enough information for ID

The mass of a single peptide provides sequence

constraints but is not sufficient for identification

Wilhelm Haas

Collision-Induced Dissociation (CID)

• Kinetic energy of parent ions is increased

• Parent ions undergo energy converting collisions

• Parent ions fall apart into product ions and neutrals

• Also referred to collision-activated dissociation (CAD)

+ +

Nathan Yates

Typical CID fragmentation pattern of peptides

H+

Nathan Yates

Combined residue mass for two amino acids Gly Ala Ser Pro Val Thr Cys Lxx Asn Asp Gln Lys Glu Met His Phe Arg Cmc Tyr Trp

AA 57 71 87 97 99 101 103 113 114 115 128 128 129 131 137 147 156 161 163 186

Gly 57 114

Ala 71 128 142

Ser 87 144 158 174

Pro 97 154 168 184 194

Val 99 156 170 186 196 198

Thr 101 158 172 188 198 200 202

Cys 103 160 174 190 200 202 204 206

Lxx 113 170 184 200 210 212 214 216 226

Asn 114 171 185 201 211 213 215 217 227 228

Asp 115 172 186 202 212 214 216 218 228 229 230

Gln 128 185 199 215 225 227 229 231 241 242 243 256

Lys 128 185 199 215 225 227 229 231 241 242 243 256 256

Glu 129 186 200 216 226 228 230 232 242 243 244 257 257 258

Met 131 188 202 218 228 230 232 234 244 245 246 259 259 260 262

His 137 194 208 224 234 236 238 240 250 251 252 265 265 266 268 274

Phe 147 204 218 234 244 246 248 250 260 261 262 275 275 276 278 284 294

Arg 156 213 227 243 253 255 257 259 269 270 271 284 284 285 287 293 303 312

Cmc 161 218 232 248 258 260 262 264 274 275 276 289 289 290 292 298 308 317 322

Tyr 163 220 234 250 260 262 264 266 276 277 278 291 291 292 294 300 310 319 324 326

Trp 186 243 257 273 283 285 287 289 299 300 301 314 314 315 317 323 333 342 347 349 372

400 600 800 1000 1200 1400 1600 1800m/z

Re

lative

Ab

undan

ce

400 600 800 1000 1200 1400 1600 1800m/z

Re

lative

Ab

undan

ce

507

200200 400400 600600 800800 10001000 12001200m/zm/z

Rela

tive A

bun

dance

Rela

tive A

bun

dance

1. MS MS/MS

2. Isolate one peptide

3. Collide with inert gas

(CID) to fragment

4. Fragment ions are

measured in MS/MS

MS/MS fragments

provide information

about the amino acid

sequence for manual

interpretation or

database searching

500 300 700 900

507.303

observed m/z

Sequencing a peptide ion using tandem MS

(MS/MS)

Wilhelm Haas

Sequencing a peptide ion using tandem MS

(MS/MS)

Wilhelm Haas

m/z

Theoretical

Spectra

Database

Experimental

Spectra

Peptide ID

Mass Spec

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib

nd_1884 #4848 RT: 28.00 AV: 1 NL: 5.36E1T: ITMS + c NSI d Full ms2 [email protected] [115.00-930.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

All rely on the database peptide sequences

(largely predicted from DNA and RNA sequences)

Search

Engine

Precursor & fragment ions

base

pea

k inte

nsity

time

organic

concentration

in mobile

phase

Separate microgram quantities of peptides on a capillary C18 column

Column I.D. = 75 μm

LC-MS/MS

We can identify 10,000 peptides in a 90 min run

HPLC

Pumps

Column Tryptic

digest

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

453.5731

583.5828

246.3161 441.1335

521.4109370.4370624.7378

568.1208

639.3729

308.8508181.3454 755.6002267.4216

667.3040147.0887823.1918

680.5607

Data Analysis

Mass

Spectrometer

A typical proteomics experiment

Wilhelm Haas

How do mass spectrometers work?

Inlet Ion

source

Mass

Analyzer Detector

Data

System

High Vacuum System

Mass analyzers use forces to manipulate ions

)( BvEzdt

vdmamF

BvEdt

vd

z

m

Time-of-flight (TOF) mass analyzer

+

+

+

+

Electric potential Drift region (flight tube)

dete

cto

r

2000 V

• Ions are formed in pulses.

• The drift region is field free.

• Measures the time for ions to reach the detector.

• Small ions reach the detector before large ones.

d

Time-of-flight (TOF) mass analyzer

+

+

+

+

Electric potential Drift region (flight tube)

dete

cto

r

2000 V

• Energy uptake is Eel=qV=ezV where e=charge/electron

• Conversion of potential energy to kinetic energy. ezV = ½ mv2

• The drift region is field free. Known distance, d.

• Measures the time for ions to reach the detector. v=d/t

• m/z=2eVv2=2eVd2/t2

d

Quadrupole mass analyzer

Oscillating electric fields, operates as a mass filter.

• Has four parallel metal rods.

• Lets one mass pass through at a time.

• Can scan through all masses or sit at one fixed mass.

mass scanning mode

(let different m/z through as function of time,

collect mass spectrum)

m1 m3 m4 m2

m3

m1

m4

m2

single mass transmission mode

let single m/z through, measure intensity

m2 m2 m2 m2 m3

m1

m4

m2

Quadrupoles have variable ion transmission

modes

Triple quadrupole MS (tandem in space)

Selected Reaction Monitoring (SRM) on a

triple quadrupole mass spectrometer

Select Fragment Select

precursor ion in Q1 precursor ion in Q2 product ion in Q3

(Collision cell containing inert gas)

Ion Trap Mass Spectrometer

accumulate ions

compress ion cloud in center

apply resonance voltage

to eject ions selectively

transfer and focus ions

Ion Trap MS process

Ionization

Precursor Ion Isolation

Collisionally Activated Dissociation

Product Ion Scanning

MS/MS on an ion trap mass spectrometer

Tandem-in-Time (allows MSn)

Time (ms)

rf

Injection

0

Eject < M

Eject > M

CAD

Daughter

Ion Scanning

How do mass spectrometers work?

Inlet Ion

source

Mass

Analyzer Detector

Data

System

High Vacuum System

hn

Laser 1. Sample is mixed with matrix (X) and

dried on plate.

2. Laser flash ionizes matrix

molecules.

3. Sample molecules (M) are ionized

by proton transfer:

XH+ + M MH+ + X.

MH+

Matrix Assisted Laser Desorption/Ionization

(MALDI)

+/- 20 kV Grid (0 V)

Sample plate

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

MH+ 225, 207

Frequently used MALDI Matrix

for proteins, large peptides

vacuum

High voltage applied

to metal sheath (~4 kV)

Sample Inlet Nozzle

(Lower Voltage)

Charged droplets

+ +

+ +

+

+

+ +

+ + + +

+ +

+ + + +

+ + +

+ + +

+ + +

+ + + +

+

+ +

+

+

+

+ + +

+ + +

+ + +

MH+

MH3+3

MH2+2

Pressure = 1 atm

Inner tube diam. = 100 um

Sample in solution

N2

N2 gas

Partial

vacuum

Electrospray Ionization (ESI) Frequently used with liquid chromatography (LC-MS)

Electrospray Ionization (ESI) Frequently used with liquid chromatography (LC-MS)

Nanospray (typically ~0.2 L/min) do not use sheath gas.

Electrospray Ionization

To MS

From µLC (M+H)+

Protonated

Molecules

Mass spectrometers are frequently named

based on their ion source & mass analyzer

Inlet Ion

source

Mass

Analyzer Detector

Data

System

High Vacuum System

MALDI

Electrospray

TOF

Quadrupole (Q)

Ion trap

Orbitrap (orbi)

Hybrid: TOF-TOF

QQQ

Q-TOF

Q-Orbi

Ion trap-Orbi

Mass Spectra

• Mass units

• Isotopes

– the good, the bad & the ugly

• Numerical value to the intrinsic property of “mass”

is based in reference to the most abundant

isotope of carbon, 12C (6 protons & 6 neutrons).

• One unit of mass is defined as a Dalton (Da).

• A Da is defined as 1/12 the mass of one 12C atom.

• Thus, one 12C atom has a mass of 12.0000… Da.

How is mass defined?

Most elements have >1 stable isotope

For example, most carbon atoms have a mass of 12 Da, but in nature,

1.11% of C atoms have an extra neutron, making their mass 13 Da.

Isotope composition of molecules depends on molecular formula and

isotope distribution of component atoms (binomial distribution).

Element Isotope Abundance

Hydrogen 1H 99.985

2H 0.015

Carbon 12C 98.890

13C 1.110

Nitrogen 14N 99.630

15N 0.370

Oxygen 16O 99.759

17O 0.037

18O 0.204

Isotope pattern (C1)+1

Monoisotopic peak, 12C1~ 99% of total (0.989)1

Isotope Pattern (C60)+1


Isotope Pattern (C300)+1


Effect of isotope abundance on mass

measurements

0 500 1000 1500 2000 2500 3000 3500 4000

mass/charge (m/z; z=1)

Inte

nsit

y

1002 1004 1006 1008

m/z

Inte

nsit

y

4014 4016 4018 4020 4022

m/z

Inte

nsit

y

2004 2006 2008 2010

m/z

Inte

nsit

y

Isotope clusters allow charge state determination

m/z

Inte

nsity 1+

1

1

1

m/z

Inte

nsity 2+

0.5

0.5

0.5

m/z

Inte

nsity 3+

0.33

0.33

0.33

David Fenyö

Resolution

Resolution = minimum peak separation, M,

which allows to distinguish two ion species

Rela

tive Inte

nsity

m/z

I I I I I

501.5 502.0 500.5 500.0 499.5

500

50 %

Resolution = M/M = 500/0.5 = 1000

M = full width at half maximum (FWHM)

R = M

M = resolving power

David Fenyö

Monoisotopic mass

Monoisotopic mass

corresponds tolowest mass peak

When the isotopes are clearly resolved the monoisotopic

mass is used as it is the most accurate measurement.

Spacing of adjacent isotope peaks (m/z, measured)

gives z as know m)

Average mass

Average mass

corresponds to the

centroid of the

unresolved peak cluster

When the isotopes are not resolved, the centroid of the

envelope corresponds to the weighted average of all the

isotope peaks in the cluster, which is the same as the

average or chemical mass.

Why we typically analyze peptides instead of

intact proteins

• Protein heterogeneity:

proteolytic processing

post translational modifications

• Stability in gas phase

• Natural isotope abundance

• Better resolution and accuracy at lower masses

Quantitative proteomics

• Label-free

• Stable isotope labeled (13C,15N, and/or 18O)

– Metabolic labeling

– Synthetic peptides and proteins

– Chemical modification

42

Quantification: Peptide concentrations are obtained by

multiplying the internal standard concentration by the

signal ratio of the analyte / internal standard

NH 2

COOH

Protein of Interest

QQQ

QQQ Detection: Selected Reaction Monitoring

provides the high sensitivity and amino acid selectivity

needed to detect peptides in complex mixtures

18 20 22 24 26 28 30 Time (min)

18 20 22 24 26 28 30 Time (min)

18 20 22 24 26 28 30 Time (min)

70

80

90

100

LC -

18 20 22 24 26 28 30 Time (min)

0

10

20

30

40

50

60

70

80

90

100 0

10

20

30

40

50

60

Rela

tive A

bundance

SRM

Analyte

Signal

= 200

Int. Std.

Signal

= 1000

NH 2

COOH

QconCAT Protein

Analyte

Peptide

AQTDIDSPQNLVTDR

IS

Peptide AQTDIDSPQNLVTDR*

Quantification of proteins by targeted

MS/MS using internal standards

Quantitative analysis using an internal standard

Sample (m/z = M) Spiked with

Internal Standard (m/z = M+i)

Inte

nsity

M

M+ i

Inte

nsity

time m/z

amol internal standard

Peak A

rea

1 10 100 1000 10000 1000000.1

1

10

100

1000

Standard (vary)Endogenous peptide (fixed)

Quantitiatve Proteomics: isobaric labeling reagents

Mol. Cell. Proteomics 3, 1154-1169(2004)

Mol. Cell. Proteomics 3, 1154-1169(2004)

mass/charge (m/z)

% I

nte

ns

ity

MS1 : multiple precursor (peptide) ions

9. 0 208. 6 408. 2 607. 8 807. 4 1007. 0

Mass ( m/ z)

7. 9E+4

0

10

20

30

40

50

60

70

80

90

100

% Intensity

4 7 0 0 M S/M S Pre c u rs o r 9 5 2 .5 6 7 Sp e c # 1 [BP = 1 1 5 .1 , 7 8 5 4 4 ]

115. 1359

175. 1362

117. 1397

116. 1317

70. 0928

87. 1082 374. 2191 521. 2928218. 1851112. 1105432. 2634 778. 4455665. 3603120. 1099 579. 3332

707. 436071. 0950 145. 1292 346. 2271229. 1705 493. 2941417. 2315129. 1252 787. 426159. 0793

328. 2076228. 1710 590. 3156516. 2964130. 1267 905. 4017832. 4753753. 334630. 0611 414. 2555 690. 3844344. 2373253. 1553 630. 3959560. 3219477. 3035124. 1270 192. 1629

Man2b1 888 -TQFSGLR- 894

9. 0 208. 6 408. 2 607. 8 807. 4 1007. 0

Mass ( m/ z)

7. 9E+4

0

10

20

30

40

50

60

70

80

90

100

% Intensity

4 7 0 0 M S/M S Pre c u rs o r 9 5 2 .5 6 7 Sp e c # 1 [BP = 1 1 5 .1 , 7 8 5 4 4 ]

115. 1359

175. 1362

117. 1397

116. 1317

70. 0928

87. 1082 374. 2191 521. 2928218. 1851112. 1105432. 2634 778. 4455665. 3603120. 1099 579. 3332

707. 436071. 0950 145. 1292 346. 2271229. 1705 493. 2941417. 2315129. 1252 787. 426159. 0793

328. 2076228. 1710 590. 3156516. 2964130. 1267 905. 4017832. 4753753. 334630. 0611 414. 2555 690. 3844344. 2373253. 1553 630. 3959560. 3219477. 3035124. 1270 192. 1629

Man2b1 888 -TQFSGLR- 894

114

Expanded scale

Reporter ion

region

115

116

117

Parting message

Progress in science depends on

new techniques,

new discoveries, and

new ideas,

probably in that order

Sydney Brenner, 20 March 1980

Biology in the 1980’s, talk at the

Friedrich Miescher Institute, Basel, Switzerland

Mass Spectrometry on the Cinco de Mayo - Rutgers...

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