MS/MS Spectral Interpretation Linda Breci Chemistry Mass Spectrometry Facility University of Arizona MS Summer Workshop.

MS/MS Spectral Interpretation

Linda BreciChemistry Mass Spectrometry Facility

University of Arizona

MS Summer Workshop

MS/MS Spectral Interpretationsmall molecule structure

Arpad SomogyiChemistry Mass Spectrometry Facility

University of Arizona

MS Summer Workshop

Session Overview

• Ways to approach predicting fragment ion formation• Fragmentation examples

– Peptides• Fragmentation mechanism• Sequence a peptide

– Flavonoids– Fatty Acids– Oligonucleotides

MS/MS Fragmentation

Few libraries, little software available for data analysis• Why?

We need useful information from MS/MS spectra

Few libraries, little software available for data analysis• Why?

For MS/MS you have at least one of each of these:

• Analyze– Q– Q-trap– linear-trap– B sectors– E sectors– FTICR– TOF

• Activate– CID– SID– SORI– IRMPD– ECD– BIRD

• Ionize– EI– CI– ESI– NSI– MALDI– FAB

You put them together like this:

* ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSector-SID-TOF * NSI-CID-Q-trap * MALDI-TOF-CID-TOF * NSI-Linear-trap-CID-FTICR * NSI-Q-trap-SID-TOF *

EI-CID-Q-trap * ESI-IRMPD-FTICR * ESI-Q-CID-Q * MALDI-TOF-CID-TOF * NSI-BIRD-FTICR * ESI-

EBSector-CID-EBSector * and on…and on…

You put them together like this:

* ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSector-SID-TOF * NSI-CID-Q-trap * MALDI-TOF-CID-TOF * NSI-Linear-trap-CID-FTICR * NSI-Q-trap-SID-TOF *

EI-CID-Q-trap * ESI-IRMPD-FTICR * ESI-Q-CID-Q * MALDI-TOF-CID-TOF * NSI-BIRD-FTICR * ESI-

EBSector-CID-EBSector * and on…and on…

– Different source designs• Example: ESI capillary temperature

– Different analyzer designs• Example: Gas pressure, length of ion path ( timeframe)

And you buy them from different manufacturers

How ions will fragment must be considered from fundamentals (rather than rules)

• Ways to approach predicting MS/MS fragment formation

• Literature– Study methods and ID’d spectra for your ion class

• Likely sites of protonation (or deprotonation)– Find proton affinities or acid strengths

• Mobility of protons– Consider the likelihood of multiple cleavage sites– Consider multiple gas-phase configurations

• Likely leaving groups

Types of ions formed

• EI (hard ionization)– M+· Radical ion– A lot of fragmentation occurs upon ionization

• CI, FAB, ESI, APCI, MALDI (soft ionization)– [M+H]+ Protonated ion– [M-H]- Deprotonated ion– [M+Na]+ and other metal cations

Today’s Topic

EI is not an MS/MS method

• Discussed Day 4

• Libraries of EI spectra are useful

• NIST/EPA/NIH Mass Spectral Library with Search http://webbook.nist.gov/chemistry/

• Libraries are not always helpful, tutorials available– http://www.chem.arizona.edu/massspec/

2 Categories of fragments from protonated or deprotonated molecules (CI, FAB, ESI, APCI, MALDI)

• Charge Remote– Fragmentation reactions uninfluenced by charge– High energy process– Charge remote references provided

• Charge Directed– Bond cleavage occurs with involvement of charge– Low energy– Most informative for many molecules

Today’s Topic

How ions will fragment must be considered from fundamentals (rather than rules)

• Literature– Study methods and ID’d spectra for your ion class

• Likely sites of protonation (or deprotonation)– Find proton affinities or acid strengths

• Mobility of protons– Consider the likelihood of multiple cleavage sites– Consider multiple gas-phase configurations

• Likely leaving groups

How ions will fragment must be considered from fundamentals (rather than rules)

• Literature– Study methods and ID’d spectra for your ion class

• Likely sites of protonation (or deprotonation)– Find proton affinities or acid strengths

• Mobility of protons– Consider the likelihood of multiple cleavage sites– Consider multiple gas-phase configurations

• Likely leaving groups

Fragmentation is a multi-step process

H2NNH

NNH

NHOH

O

O

O

O

O

H

Step #1: Create Ions (add 1 or more protons)

ELECTROSPRAY

Fragmentation is a multi-step process

H2NNH

NNH

NHOH

O

O

O

O

O

H

H2NNH

NNH

NHOH

O

O

O

O

O

H

H2NNH

NNH

NHOH

O

O

O

O

O

H

H2NNH

NNH

NHOH

O

O

O

O

O

H

Step #1: Create Ions (add 1 or more protons)

Step #2: Add energy (activation)

SID

CID

ELECTROSPRAY

H2NNH

NNH

NHOH

O

O

O

O

O

H

y3

b2

a2

H2NNH

NNH

NHOH

O

O

O

O

O

R1

R2

R3

R4

R5

y2

b3

a3

Step #3: Charge Directed Cleavage

Fragmentation is a multi-step process

Neutral + Fragment ion

What are the likely sites of proton location?

Model possible sites of proton location(or loss of H) in Serine

H2N CH C

CH2

OH

O

OH

M + H → [M+H]+ Hrxn = -PA (M)

M → [M - H]- + H+ Hrxn = Hacid (M)

Model possible sites of proton location(or loss of H) in Serine

H2N CH C

CH2

OH

O

OH

Model with CH3NH2

(methyl amine)

M + H → [M+H]+ Hrxn = -PA (M)

M → [M - H]- + H+ Hrxn = Hacid (M)

Model possible sites of proton location(or loss of H) in Serine

H2N CH C

CH2

OH

O

OH

Model with CH3NH2

(methyl amine)

M + H → [M+H]+ Hrxn = -PA (M)

M → [M - H]- + H+ Hrxn = Hacid (M)

Ref: NIST

PA H acid 402.0214.9methyl amine

Model possible sites of proton location(or loss of H) in Serine

H2N CH C

CH2

OH

O

OH

Model with CH3COOH(acetic acid)

Model with CH3NH2

(methyl amine)

M + H → [M+H]+ Hrxn = -PA (M)

M → [M - H]- + H+ Hrxn = Hacid (M)

Ref: NIST

PA H acid

348.1187.3acetic acid

402.0214.9methyl amine

Model possible sites of proton location(or loss of H) in Serine

H2N CH C

CH2

OH

O

OH

Model with CH3COOH(acetic acid)

Model with CH3NH2

(methyl amine)

Model with CH3OH(methanol)

M + H → [M+H]+ Hrxn = -PA (M)

M → [M - H]- + H+ Hrxn = Hacid (M)

Ref: NIST

PA H acid

382.0180.3methanol

348.1187.3acetic acid

402.0214.9methyl amine

Model possible sites of proton location(or loss of H) in Serine

H2N CH C

CH2

OH

O

OH

Model with CH3COOH(acetic acid)

Model with CH3NH2

(methyl amine)

Model with CH3OH(methanol)

M + H → [M+H]+ Hrxn = -PA (M)

M → [M - H]- + H+ Hrxn = Hacid (M)

Ref: NIST

PA H acid

382.0180.3methanol

348.1187.3acetic acid

402.0214.9methyl amine

Sites of Likelyprotonation: NH2 > COOH > OHdeprotonation: COOH > OH > NH2

How ions will fragment must be considered from fundamentals (rather than rules)

• Literature– Study methods and ID’d spectra for your ion class

• Likely sites of protonation (or deprotonation)– Find proton affinities or acid strengths

• Mobility of protons– Consider the likelihood of multiple cleavage sites– Consider multiple gas-phase configurations

• Likely leaving groups

Proton mobility

• Intramolecular proton transfer influences– number of site-directed fragmentations– amount of energy required for fragmentation

• Intramolecular proton transfer affected by– site basicity– gas-phase configuration

• Examples that follow:– Spectra of increasingly basic peptides– Overview chart demonstrating proton mobility (or lack of)– Spectra of peptide conformers

Ref: Gu, 1999

CompareGas Phase Basicity

Arg (R):240.6 kcal/mol

Lys (K):227.3 kcal/mol

His (H):227.3 kcal/mol

50 eV(SID)

40 eV(SID)

40 eV(SID)

Pairwise bond cleavage between amino acids (Xxx-Zzz)

Z z z

1

2

3

4

5

6

7

8

9

10 Most Abundant

LeastAbundant

Peptides with more basic Arg (R) vs. Lys (K)

.............R 1+ .............K

Prediction based on model peptides: Selective Cleavage at Asp-Xxx will depend on number of

“Mobile” Protons

H H

or Arg or Lys

His Arg (Lys)Asp

H HArg (Lys)

Asp

H HArg (Lys)

Asp

Huang, Wysocki, Tabb, Yates Int. J. Mass Spectrom. 219, (1), 233-244, 2002

Peptides with basic Arg (R) 1 proton vs. 2 protons

1+ .............R 2+

H2N CH C

CH3

O

HN CH C

CH3

O

N

C

O

HN CH C

CH3

O

HN CH C

CH3

OH

O

Gas-phase conformation influences MS-MS spectra observed

Ala-Ala-Pro-Ala-Ala

Most Natural occurring amino acids have L configuration at the chiral center (stereospecific biosynthesis)

Calculated structure of [AAPAA + H]+

Many sites of possible interaction

No solvent in the gas phase!

Gas-phase confirmation can influence MS-MS spectra observed

Peptides containing proline stereoisomers fragment differently

All L-amino acidsAll L-amino acids

except central residueAVDPLG

0 100 200 300 400 5000

20

40

60

80

100

MH+

PLb

3

y3

a4

b4

SID spectra of [AV(D)

PLG+H]1+ (29eV)

m/z

0 100 200 300 400 5000

50

100

150

200

250

300

350

PL

y3

MH+

b4

SID spectra of [AV(L)

PLG+H]1+ (29eV)

m/z

Gas-phase confirmation can influence MS-MS spectra observed

Peptides containing proline stereoisomers fragment differently

All L-amino acidsAll L-amino acids

except central residueAVDPLG

0 100 200 300 400 5000

20

40

60

80

100

MH+

PLb

3

y3

a4

b4

SID spectra of [AV(D)

PLG+H]1+ (29eV)

m/z

0 100 200 300 400 5000

50

100

150

200

250

300

350

PL

y3

MH+

b4

SID spectra of [AV(L)

PLG+H]1+ (29eV)

m/z

Gas-phase confirmation can influence MS-MS spectra observed

Peptides containing proline stereoisomers fragment differently

All L-amino acidsAll L-amino acids

except central residueAVDPLG

0 100 200 300 400 5000

20

40

60

80

100

MH+

PLb

3

y3

a4

b4

SID spectra of [AV(D)

PLG+H]1+ (29eV)

m/z

0 100 200 300 400 5000

50

100

150

200

250

300

350

PL

y3

MH+

b4

SID spectra of [AV(L)

PLG+H]1+ (29eV)

m/z

Statistical analysis of cleavage at the Xxx-Pro bond

Val HisAsp Ile Leu Lys Glu Phe Tyr Ala Gln Thr Asn Arg Trp Ser Gly Pro

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Rel

ativ

e In

tens

ity [

(a+

b+y)

Xxx

-Pro /

(a+

b+y)

all]

Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971

Statistical analysis of cleavage at the Xxx-Pro bond

Val HisAsp Ile Leu Lys Glu Phe Tyr Ala Gln Thr Asn Arg Trp Ser Gly Pro

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Rel

ativ

e In

tens

ity [

(a+

b+y)

Xxx

-Pro /

(a+

b+y)

all]

Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971

Asp, His = Selective cleavage residuesVal, Ile, Leu = Bulky aliphatic side chains

How ions will fragment must be considered from fundamentals (rather than rules)

• Literature– Study methods and ID’d spectra for your ion class

• Likely sites of protonation (or deprotonation)– Find proton affinities or acid strengths

• Mobility of protons– Consider the likelihood of multiple cleavage sites– Consider multiple gas-phase configurations

• Likely leaving groups

Likely Leaving Groups

• Bond cleavage is dependent on various factors including:– Leaving Groups– Neighboring group participation reactions– Intermediates (ion-neutral complex)

• For [M+H]+ ions the leaving group is a neutral– lower methyl cation affinity is one measure of likelihood– Compilations available in the literature– Related to proton affinity

Ref: Bartmess, 1989

(kcal/mol)

Proton Affinity vs. Methyl Cation Affinity

Ref: Bartmess, 1989

Some fragmentation studies & basics

• Few examples from literature – Cannot talk about all classes of compounds– These examples suggest problem solving approaches

• Examples:– Peptides

• Fragmentation mechanism

• Sequence a peptide

– Flavonoids– Fatty Acids– Oligonucleotides

Peptides

• Product ion spectra contain many types of fragment ions– charge directed– charge remote– internal fragments– immonium ions

• Important for sequencing– amino acid determined from mass between peaks in spectrum– “y” ions series – “b” ions series– immonium ions (identify amino acids in the peptide)– “a” ions (confirm “b” ion after a loss of CO, 28 amu)

• Presented here:– peptide fragment ions– a mechanism for fragment ion formation – a peptide to sequence

Peptide bond fragment ions

Peptide fragment ions

H2N CH C

H

O

HN CH C

H

O

HN CH C

H

O

HN CH C

H

OH

O

CH

R

H2N C

N

CH

R'O

H

Internal immonium ion Amino acid immonium ion

a2

b2

c2

x2

y2

z2

H2N CH

R

Protonation occurs at amide oxygen or nitrogen

Ref: Yalcin, 1996

(Peptide) CH C

R1

O

N CH C N CH C

R3

(Peptide)

O

O

R2

H H

H

Protonation occurs at amide oxygen or nitrogen

Ref: Wysocki, 2000

(Peptide) CH C

R1

O

N CH C N CH C

R3

(Peptide)

O

O

R2

H H

H

A mechanism of peptide fragmentation

(Peptide) CH C

R1

O

N CH C N CH C

R3

(Peptide)

O

O

R2

H H

H

(1) positive charge(2) Nucleophilic attack

Ref: Wysocki, 2000

A mechanism of peptide fragmentation

(3) cyclic intermediate

(Peptide) CH C

R1

O

N CH C N CH C

R3

(Peptide)

O

O

R2

H H

H

(1) positive charge(2) Nucleophilic attack

(Peptide) CH

R1O

N R2

OH

H

HN CH C

CH3

(Peptide)

O

Ref: Wysocki, 2000

A logical mechanism of peptide fragmentation

(4) Rearrangement

(Peptide) CH

R1O

N R2

OH

H

HN CH C

CH3

(Peptide)

O

(Peptide) CH

R1O

HN R2

OH

HN CH C

CH3

(Peptide)

O

(3) cyclic intermediate

Ref: Wysocki, 2000

A logical mechanism of peptide fragmentation

(Peptide) CH

R1O

HN R2

O

H2N CH C

CH3

(Peptide)

O

H2N CH C

R3

(Peptide)

O

(Peptide) CH

R1O

N R2

O

H

b oxazolone ion neutral

+

Ref: Wysocki, 2000

A logical mechanism of peptide fragmentation

oxazolone neutral(or other structure)

y ion

+

(Peptide) CH

R1O

HN R2

O

H2N CH C

CH3

(Peptide)

O

(Peptide) CH

R1O

N R2

O

H2N CH C

R3

(Peptide)

O

H

Ref: Wysocki, 2000

Peptide bond fragment ions

Peptide fragment ions

H2N CH C

H

O

HN CH C

H

O

HN CH C

H

O

HN CH C

H

OH

O

CH

R

H2N C

N

CH

R'O

H

Internal immonium ion Amino acid immonium ion

a2

b2

c2

x2

y2

z2

H2N CH

R

Peptide Sequencing

mass amino acid   

Alanine ALA A 71.09

Arginine ARG R 156.19

Aspartic Acid ASP D 115.09

Asparagine ASN N 114.11

Cysteine CYS C 103.15

Glutamic Acid GLU E 129.12

Glutamine GLN Q 128.14

Glycine GLY G 57.05

Histidine HIS H 137.14

Isoleucine ILE I 113.16

Leucine LEU L 113.16

Lysine LYS K 128.17

Methionine MET M 131.19

Phenylalanine PHE F 147.18

Proline PRO P 97.12

Serine SER S 87.08

Threonine THR T 101.11

Tryptophan TRP W 186.12

Tyrosine TYR Y 163.18

Valine VAL V 99.14

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp

LEARNING CHECK

Peptide Sequencing Exercise

Ion Currentover 60 min

MS

MS/MS

Peptide precursor ions observed by MS

MH+

m/z = 1141.3

[M+ 2H]2+

m/z = 571.2

calculation of MH+

571.2 m/z measured x 2 1,142.4 [M+2H] - 1.0 1,141.4 [M+H]

MS-MS of 571.2

895.25

Peptide Sequencing

mass amino acid   

Alanine ALA A 71.09

Arginine ARG R 156.19

Aspartic Acid ASP D 115.09

Asparagine ASN N 114.11

Cysteine CYS C 103.15

Glutamic Acid GLU E 129.12

Glutamine GLN Q 128.14

Glycine GLY G 57.05

Histidine HIS H 137.14

Isoleucine ILE I 113.16

Leucine LEU L 113.16

Lysine LYS K 128.17

Methionine MET M 131.19

Phenylalanine PHE F 147.18

Proline PRO P 97.12

Serine SER S 87.08

Threonine THR T 101.11

Tryptophan TRP W 186.12

Tyrosine TYR Y 163.18

Valine VAL V 99.14

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp

895.25

895.25

895.25

FPhe

895.25

FPhe

GGly

895.25

FPhe

GGly

TThr

895.25

FPhe

GGly

TThr

DAsp

895.25

FPhe

GGly

TThr

DAsp

MMet

895.25

FPhe

GGly

TThr

DAsp

MMet

DAsp

895.25

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

Build the peptide:selected peptide = 1141.4Estimate the number of amino acids

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

Possibly 10 amino acidsConsider a y-ion series

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

1141.4 selected MH+

y series ions

1141

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

1141.4 selected MH+

1042.6 Largest fragment observedy series ions

11411042

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

1141.4 selected MH+

1042.6 Largest fragment observed 98.8 differenceIs there an amino acid with that mass?

y series ions

11411042

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

99 = ValineThe missing amino acidWhat is the next mass observed?y series ions

11411042

V

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

895

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

If this is a y-ion series:262 = smallest ion in the serieswhat does it represent?

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

All amino acids in table are peptide bond to peptide bond

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

We’re missing one N-terminal hydrogen

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp

H

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

We’re missing one C-terminal OH Group

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp

OH

H

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

And the ionizing proton Total = 19 amu

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp

OH

H

H+

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

262 = smallest identified fragment- 19 = mass of H + OH + H243 = mass of missing amino acids What amino acids?

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

262 = smallest identified fragment- 19 = mass of H + OH + H243 = mass of missing amino acids What amino acids?

Hint:Tryptic!

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N

262895

87 = Serine156 = Arginine243 19 = mass of H + OH + H262

115 = Aspartic Acid128 = Lysine243 19 = mass of H + OH + H262

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

895.25

__ __ __ __ __ __ __ __ __ __

y series ions

11411042

V F G T D M D N S R

262895

87 = Serine156 = Arginine243 19 = mass of H + OH + H262

Some fragmentation studies & basics

• Few examples from literature – Cannot talk about all classes of compounds– These examples suggest problem solving approaches

• Examples:– Peptides

• Fragmentation mechanism

• Sequence a peptide

– Flavonoids– Fatty Acids– Oligonucleotides

Flavonoids

• Common secondary plant metabolite– Including flavonoid aglycones, O-glycosides, C-glycosides (arrows)

• Need reliable methodology for analysis

Ref: Cuyckens 2004

Flavonoids

• Group classification, chalcone aglycones, etc.

Ref: Cuyckens 2004

Flavonoids

• Group classification, chalcone aglycones, etc. • Reported structures

400

450

350

300

19

250

Ref: Cuyckens 2004

Ion nomenclature for flavonoid glycosides(apigenin 7-O-rutinoside illustrated)

nomenclature suggested by Ma, 1997 and Domon,1988

Ion nomenclature for flavonoid glycosides(apigenin 7-O-rutinoside illustrated)

A and B ions (retro-Diels-Alder reactions) are most diagnostic: - provide number and type of substituents in A & B ring

Low-energy CID (Fab-Magnetic sector-Quadrupole)

luteolin kempferol

flavonetypical1,3B+

0,4B+

0,4B+-H2O

flavonoltypical0,2A+

0,2A+-CO1,4A++2H1,3B+-2H

Ref: Ma, 1997

Low-energy CID (Fab-Magnetic sector-Quadrupole)

luteolin (flavone) kempferol (flavonol)

Low-energy CID (Fab-Magnetic sector-Quadrupole)

kempferol (flavonol)luteolin (flavone)

Some fragmentation studies & basics

• Few examples from literature – Cannot talk about all classes of compounds– These examples suggest problem solving approaches

• Examples:– Peptides

• Fragmentation mechanism

• Sequence a peptide

– Flavonoids– Fatty Acids– Oligonucleotides

Fatty Acids

• Fragments formed by cleavage at alkyl bond can occur by charge remote fragmentation (generally at higher energies)– High Energy: Sector (KeV)– Low Energy: QQQ, Qtrap, FTICR– Intermediate Energy: Sector hybrids, TOF/TOF (collision gas, i.e. Xe)

• Homolytic bond-fragmentation mechanism (C--C → C- + -C radicals)

• 1,4-H2 elimination mechanism (Jensen, Tomer, Gross, 1985)

– X = O- or OLi2+

Ref: Jensen, 1985

Fatty Acids

• H-atom cleavage CRF mechanism (Claeys & Van den Heuvel, 1994)– X = OLi2+ or OBuLi+

Ref: Claeys, 1994

Stearic acid (ESI-Sector-OATOF, 400eV collision, Xe)

Ref: Griffiths, 2003

Oleic acid (ESI-Sector-OATOF, 400eV collision, Xe)

Ref: Griffiths, 2003

docosahexaenoic acid ANSA derivative (Sector, 400eV collision, Xe)

Ref: Griffiths, 2003

Gaps due to double bond

docosahexaenoic acid ANSA derivative (QQQ, 30 eV collision, Ar)

Ref: Griffiths, 2003

Gaps due to double bond

Some fragmentation studies & basics

• Few examples from literature – Cannot talk about all classes of compounds– These examples suggest problem solving approaches

• Examples:– Peptides

• Fragmentation mechanism

• Sequence a peptide

– Flavonoids– Fatty Acids– Oligonucleotides

Oligonucleotides

• McLuckey Nomenclature for multiply charged anions– Gentle collisional activation = base loss– Moderate conditions = consecutive fragmentations

Ref: McLuckey, 1993

Comparison of activation methodsCAD (CID) vs. IRMPD (Quadrupole Ion trap)

Ref: Keller, 2004

IRMPD:Low mass observed- PO3

-1

-base anions-Complete coverage

Parent-3

Comparison of activation methodsCAD (CID) vs. IRMPD (Quadrupole Ion trap)

Ref: Keller, 2004

CAD:Loss of base-provides little info -leads to backbone cleavagesComplete coverage

IRMPD:Low mass observed- PO3

-1

-base anions-Complete coverage

Parent-3

Comparison of activation methodsCAD (CID) vs. IRMPD (Quadrupole Ion trap)

Ref: Keller, 2004

CAD:Loss of base-provides little info -leads to backbone cleavagesComplete coverage

IRMPD:Low mass observed- PO3

-1

-base anions-Complete coverage

Parent-3

Steps for interpretation of oligonucleotide mass spectra for determination of sequence

Ref: Ni, 1996

Steps for interpretation of oligonucleotide mass spectra for determination of sequence

Ref: Ni, 1996

Comments on steps to interpretation

Ref: Ni, 1996

General MS/MS

NIST Chemistry WebBook http://webbook.nist.gov/chemistry/

Rossi, D.T., Sinz, M.W., Mass Spectrometry in Drug Discovery, 2002, Marcel Dekker, Inc., New York, NY.

Bartmess, J.E., Gas-Phase Equilibrium Affinity Scales and Chemical Ionization Mass-Spectrometry, Mass Spec. Reviews,1989, 8:297-343. (Affinity Tables)

McCloskey, J.A., Ed., Tandem Mass Spectrometry, Methods in Enzymology, 1990, Vol 193, Academic Press, N.Y.

Peptides

Gu, C., Somogyi, A., Wysocki, V.H., Medzihradszky, K.F., Fragmentation of protonated oligopeptides XLDVLQ (X=L, H, K or R) by surface induced dissociation: additional evidence for the ‘mobile proton’ model., Analytica Chem. Acta, 1999, 397:247-256

Yalcin, T., Csizmadia, I.G., Peterson, M.R., Harrison, The Structure and Fragmentation of Bn (n ≥ 3) Ions in Peptide Spectra., A.G., J. Am. Soc. Mass Spectrom., 1996, 6, 1164-1174.

Wysocki, V.H., Tsaprailis, G., Smith, L., Breci, L., Mobile and localized protons: a framework for understanding peptide dissociation, J. Mass Spectrom., 2000, 35, 1399-1406.

Flavonoids

Cuyckens, F., Claeys, M., Mass spectrometry in the structural analysis of flavonoids, J. Mass Spectrom. 2004; 39: 1–15.

Ma, Y.L., Li, Q.M., Van den Heuvel, H., Claeys, M., Characterization of flavone and flavonol aglycones by collision-induced dissociation tandem mass spectrometry, RCMS, 1997, 11: 1357.

Suggested Reading List & References

Domon, B., Costello, C.E., A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J., 1988, 5:397.

Fatty Acids & Charge Remote

Griffiths, W., Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids, Mass Spec. Reviews, 2003, 22, 81-152.

Jensen, N.J., Tomer, K.B., Gross, M.L., Gas phase ion decomposition occurring remote to a charge site, J.Am.Chem.Soc., 1985, 107:1863-1868.

Claeys M., Van den Heuvel, H., Radical processes in remote charge fragmentations of lithium cationized long-chain alkenyl and alkadienyl salicylic acids, Biol. Mass Spec., 1994, 23:20-26.

Gross, M.L., Charge-remote fragmentations – method, mechanism and applications, Int.J.Mass Spec.Ion Process., 1992, 118: 137-165.

Wysocki, V.H., Ross, M.M., Charge-remote fragmentation of gas-phase ions – mechanistic and energetic considerations in the dissociation of long-chain functionalized alkanes and alkenes, Int.J.Mass Spec.Ion Process, 1991, 179-211.

Oligonucleotides

McLuckey, S.A., Habibi-Goudarzi, S., Decompositions of multiply Charged Oligonucleotide Anions, J.Am.Chem.Soc., 1993, 115:12085-12095.

Keller, K.M., Brodbelt, J.S., Collisionally activated dissociation and infrared multiphoton dissociation of oligonucleotides in a quadrupole ion trap, Anal.Chem., 2004, 326:200-210.

Ni, J.S., Pomerantz, S.C., Rozenski, J., Zhang, Y.H., McCloskey, J.A., Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry, Anal.Chem., 1996, 68:1989-1999.

Suggested Reading List & References (2)