Peptide ion fragmentation in

mass spectrometry
p y
Matthew B. Renfrow
renfrow@uab.edu
6-4681

S. Barnes & M. Renfrow 1/14/11

 Where we are so far
• We’ve discussed the nature of the problem, how
we might attack it and what we believe in
• Matt
M tt R
Renfrow
f h
has ttold
ld you
– how (remarkably) we get peptide and protein molecular
ions into gas phase
– the importance of isotopes in mass spectrometry
– how we measure the m/z values of the ions
• We also talked about:
– how to measure the molecular weight of a protein
– How to fragment a protein into smaller pieces to get a
peptide mass fingerprint and hence “identify” it

S. Barnes & M. Renfrow 1/14/11

 Lecture goals
• Value of fragmentation in determining
structure
• How peptides fragment
– Interpreting the tandem mass spectrum
• Automating identification of peptides from
their fragment ions
– pros and cons
• Controlling fragmentation
– Choice of ionization and fragmentation
methods

S. Barnes & M. Renfrow 1/14/11

 Why ion fragmentation
provides useful information
• Compounds can have the same empirical
formula, i.e., the same molecular weight or m/z,
b t be
but b different
diff t chemically.
h i ll
• Breaking them into parts (fragmenting them)
h l to
helps t identify
id tif what
h t they
th are.
• Each of the following peptides gives rise to
exactly
l the
h same m/z / for
f the
h [M+2H]
[M 2H]2+
2 ion
i
– NH2VFAQHLK-COOH NH2VAFQHLK-COOH
– NH2VFQHALK-COOH
VFQHALK COOH NH2VHLAFQK
VHLAFQK-COOH
COOH

• In proteomics we want to distinguish these

peptides
S. Barnes & M. Renfrow 1/14/11
 What is MS
MS-MS
MS (tandem
mass spectrometry)?
gas

Molecular ions Analyzer 1 selected ion Analyzer 2

Collision induced dissociation

Collision-induced
S. Barnes & M. Renfrow 1/14/11
 Fragmenting
g g a peptide
p p
x2 y2 z2

NH3+-CHR
CHR1-CO-NH-CHR
CO NH CHR2-CO-NH-CHR
CO NH CHR3-CO-NH-CHR
CO NH CHR4-COOH
COOH

R1 O R2 a2 b2 c2 R3 O R4
| || | | || |
H2N--C--C--N =C
+
a2 +O=C--HN--C--C--N--C--COOH x2
| | | | | |
H H H H H H

R1 O R2
| || | R3 O R4
H2N--C--C--N--C--C=O+ b2 | || |
| | | H3N --C--C--N--C--COOH
+ y2
H H H | | |
H H H

R1 O R2 O R3 O R4
| || | || | || |
H2N--C--C--N--C--C--NH3+ c2 +C--C--N--C--COOH z2
| | | | | |
H H H H H H
Adapted from http://www.matrixscience.com/help/fragmentation_help.html

S. Barnes & M. Renfrow 1/14/11

 Calculating expected b- and y-ion
fragments
Alanine 71.037 Leucine 113.084
Arginine 156.101 Lysine 128.094
Asparagine 114.043 Methionine 131.040
Aspartic acid 115.027 Phenylalanine 147.068
Cysteine 103.009 Proline 97.053
Glutamic acid 129.043 Serine 87.032
Glutamine 128.058 Threonine 101.048
Glycine 57 021
57.021 Tryptophan 186 079
186.079
Histidine 137.059 Tyrosine 163.063
Isoleucine 113.084 Valine 99.068

bn = [residue masses + 1] - these come from the N-terminus

yn = [residue masses + H2O + 1] = these come from the C-terminus

b3 = ADG,
ADG 71.04+115.03+57.02+1=
71 04+115 03+57 02+1= 244.09
244 09
ADGTWLEVR
y3 = EVR, 129.04+99.07+156.10+18+1= 403.21

S. Barnes & M. Renfrow 1/14/11

 Identification of daughter ions and
peptide sequence y10
1100.61
100

y9
987.52

b3
375.21 Charge state? =147.08
% b2 y8
262.12 916.49

parent
234.13 681.45
y11
1247.69
y1 b8
b4
175 12
175.12 446 23
446.23 y4 y5 y6 y7
875.42
487.32 602.32 730.43 859.45
y2
274.19

0 mass
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

S. Barnes & M. Renfrow 1/14/11

 What’s
What s in a peptide MSMS
spectrum?
• In most cases, some, but rarely all, of the
th
theoretic
ti b-
b andd y-ions
i are observed
b d
• Besides b- and y-ions, other types of
fragmentation can occur to form an and xn
ions, as well as also losing CO, NH3 and
H2O groups
g reactions can occur at
• Internal cleavage
acidic (Asp - Glu) residue sites

S. Barnes & M. Renfrow 1/14/11

 GalNAc

FT-ICR-MS
Gal
Gal

1400 1450 1500 1550 1600 1650 m/z

quadrupole
+ SWIFT
Renfrow M B et al. J. Biol. Chem. 2005;280:19136-19145
isolated

1400 1450 1500 1550 1600 1650 m/z

GalNAc Gal GalNAc
IRMPD
Gal GalNAc Gal MS / MS

1400 1450 1500 1550 1600 1650 m/z

 Identifying a peptide by de
novo sequencing
• Take the partial sequence that can be
identified manually and submit it to
PROWL (http://prowl.rockefeller.edu/) -
click on PROTEININFO and enter
sequence - select all species
• Use suggested sequences to fill in the
gaps and then check all theoretical ions
usingg MS-Product at
http://prospector.ucsf.edu/prospector/cgi-
bin/msform.cgi?form=msproduct

S. Barnes & M. Renfrow 1/14/11

 CID Peptide
Fragmentation TFLVWVNEEDHLR

Inert Gas
(He)
102 T FLVWVNEEDHLR 1556
249 TF LVWVNEEDHLR 1409
362 TFL VWVNEEDHLR 1296
N terminal 461
N-terminal TFLV WVNEEDHLR 1197
C-terminal
b ions 647 TFLVW VNEEDHLR 1911 y ions
m/z 746 TFLVWV NEEDHLR 912 m/z
860 TFLVWVN EEDHLR 798
989 TFLVWVNE EDHLR 669
The ion’s mass
which would be
118 TFLVWVNEE DHLR 540
affected by a 1233 TFLVWVNEED HLR 425
modified H? 1370 TFLVWVNEEDH LR 288
1483 TFLVWVNEEDHL R 175
S.Eliuk & H. Kim 1/14/2011
Fragmentation by CID
(Collision Induced Dissociation)

http://www.ionsource.com/tutorial/DeNovo/nomenclature.htm

The most common fragments observed with ion

trap triple quadrupole
trap, quadrupole, and QTOF mass
spectrometers
S.Eliuk & H. Kim 1/14/2011
4HNE Michael Adduction of H66
*
b5

TLDDV I QTGVDNPGHPY
b6 b7 b8 b10 b11 b12 b13 b14 b15

LTQ-MS/MS y12 y11 y10 y9 y8 y7 y6 y5 y4 y3

*
y6 + *
y8+ *
y11+
b11+
*
y4+ b10 +

b5+ b6+
*
b8+ y10+ * b13+
y5+
* y*
y7 +
+ y*
* +
b15+
9 12
b12+
b14+
b7+

0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 0 0 1 6 0 0 1 8 0 0 2 0 0 0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
S.Eliuk & H. Kim 1/14/2011
S. Barnes 1/15/10
S. Barnes 1/15/10
 Other ions observed in CID peptide
fragmentation

Immonium and Related Ions

84.08 70.07
87.06 120.08 86.10 --- --- 102.05 101.11 88.04 87.06 72.08 72.08 87.09
129.10 100.09
112.09
N-terminal ions
a-NH3 ions --- 217.10 330.18 401.22 458.24 587.28 715.38 830.40 944.45 1043.52 1142.58 ---
a ions --- 234.12 347.21 418.24 475.27 604.31 732.40 847.43 961.47 1060.54 1159.61 ---
b-NH3 ions --- 245.09 358.18 429.21 486.23 615.28 743.37 858.40 972.44 1071.51 1170.58 ---
b-H2O ions --- --- --- --- --- 614.29 742.39 857.42 971.46 1070.53 1169.59 ---
b ions --- 262.12 375.20 446.24 503.26 632.30 760.40 875.43 989.47 1088.54 1187.61 ---

1 2 3 4 5 6 7 8 9 10 11 12
H- N F L A G E K D N V V R

y ions --- 1247.67 1100.61 987.52 916.48 859.46 730.42 602.33 487.30 373.26 274.19 175.12
y-NH3 ions --- 1230.65 1083.58 970.50 899.46 842.44 713.39 585.30 470.27 356.23 257.16 158.09

y-H2O ions --- 1229.66 1082.60 969.51 898.47 841.45 712.41 584.32- --- --- -- ---

S. Barnes & M. Renfrow 1/14/11

 Identification of daughter ions and
peptide sequence y10
1100.61
100 b ions 262 375 446 503 632 760 875 989 1088 1187 1343
N F L A G E K D N V V R
y ions 1361 1247 1100 987 916 859 730 602 487 373 274 175
y9
987.52

b3
375.21

% b2 y8
262.12 916.49

parent
234.13 681.45
y11
1247.69
y1 b8
b4
175 12
175.12 446 23
446.23 y4 y5 y6 y7
875.42
487.32 602.32 730.43 859.45
y2
274.19

0 mass
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

S. Barnes & M. Renfrow 1/14/11

 Issues in MS-MS experiment
• At any one moment, several peptides may
be co-eluting
• Data-dependent operation:
– The most intense peptide molecular ion is
selected first (must exceed an initial
threshold value) threshold
– A 2-3 Da window is used (to maximize the
signal)
– The ion must be in 2+ or 3+ state
– Since the ion trap scan of the fragment
ions takes ~ 1 sec, only the most intense
i
ions will
ill be
b measured d
– However, can use an exclusion list on a
subsequent run to study minor ions

S. Barnes & M. Renfrow 1/14/11

 VL
CL

VH
NeuAc
C1
C C α2,3
23
Gal
C1
C C β1 3
β1,3
VH

GalNAc NeuAc
VL
CL
α2 6
α2,6
Hinge Fc
Fab
region Ser/Thr

Trypsin Pepsin

CHVK HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL SLHR

(CHO) CHO CHO CHO (CHO)

CH1 CH2
-Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys-
225 228 230 232 236
 ESI FT-ICR positive ion mass spectra of isolated IgA1 HR peptide.

R f
Renfrow M B ett al.
l JJ. Bi
Biol.
l Ch
Chem. 2005
2005;280:19136-19145
280 19136 19145

©2005 by American Society for Biochemistry and Molecular Biology

IgA1 hinge region O-glycosylation
neuraminidase + O-glycanase treated
GalNac

de-glycosylated
de glycosylated HR peptide

1000 1100 1200 1300 1400 1500 1600 1700 m/z

neuraminidase treated GalNac

Gal

Gal

1000 1100 1200 1300 1400 1500 1600 1700 m/z

IgA1 hinge region O-glycosylation

VTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL
[M + 3H]3+
y273+ y282+
IRMPD y293+ ~x5

y132+/ b7 y242+
~x5

y17 2+ y262+
y102+ 2+ y272+ [M + 2H]2+
y16
y3 y212+
b3 b5 y142+ y222+
2

y292+

300 500 700 900 1100 1300 1500 m/z

 Let’s take a closer look at
fragmentation

b3 ion - oxazalone
no b1 ion

S. Barnes & M. Renfrow 1/14/11

Wysocki et al. 2005
Other amino acid fragment ions

S. Barnes & M. Renfrow 1/14/11

Wysocki et al. 2005
Detecting posttranslational
modifications (PTMs) by MS
• A key issue is that the energy of ionization or the collisional
process should not exceed the dissociational energy gy of the
PTM

• MALDI-TOF
MALDI TOF MS with a N2 laser causes fragmentation of a
nitrated tyrosine residue
– Use ESI to make the molecular ion
– Go to another laser wavelength (YAG laser at 355 nm or IR)

• O-glucosyl and phospho groups fragment more easily than

the peptide to which they are attached
– Use electron capture dissociation

S. Barnes & M. Renfrow 1/14/11

IgA1 hinge region O-glycosylation
VTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL

= GalNAc-Gal

= GalNAc
GalNac

Gal

Gal

1400 1450 1500 1550 1600 1650 1700 1750 m/z

 Types of fragmentation (1)
• Collision-induced dissociation (CID)
– Also called CAD (collision-activated dissociation)
– Multiply
p y charged
g peptide
p p ions are isolated byy an m/z
based filter
– Selected ions are accelerated into a field of inert gas
(He N2, Ar,
(He, Ar Xe) at moderate pressure
– The energy gained in collision events increases
vibrational and stretching modes of the peptide
backbone (and anything attached to it!)
– The increased motion of the energized peptide causes
breaks that occur typically at the peptide bond
• Side chain groups can also be broken, some times more
easily than the peptide chain

S. Barnes & M. Renfrow 1/14/11

Fragmentation
g of nitrated peptides
p p
in MALDI-TOF experiment

S. Barnes & M. Renfrow 1/14/11

ESI-tandem MS of a nitrated peptide

S. Barnes & M. Renfrow 1/14/11

 aa658-668 RNSILTETLHR Unmodified m/z 447.37(3S.+)Barnes 1/15/10
1339.09 Da

448.7

b-ions -- -- 358 471 584 685 814 915 -- -- --

R N S I L T E T L H R
-- -- -- -- 869 756 655 526 425 312 175 y-ions

471.3(b4)

526.6(y4)
584.6(b5) 756.5(y6)
425.5(y3)
685 6(b6)
685.6(b
447.7

312.0(y2) 358.3(b3) 655.5(y5)

330.5
869.6(y7)
556.8
380.2
667.6 814.6(b7) 915.6(b8)
637.6 738.6
175.2(y1) 509.5

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150
m/z, amu
S. Barnes 1/15/10
 aa658-668 RNsILTETLHR Phosphorylated m/z 474.00(3+) 1419.00 Daltons
474.2 (parent)
453.4(b4-98)

b-ions -- 271 340 453 566 -- -- -- -- -- --

R N s I L T E T L H R
-- -- -- -- -- 756 655 526 425 312 175 y-ions

340.3(b3-98) 526.4(y4)
175.1(y1)
566.5(b5-98)
98)
271.2(b2)
425.4(y3) 756.5(y6)
655.4(y5)

312.3(y2)
187.2 664.2
130.1
157.1
379.2 426.5
155.2170.3 441.7
213.1263.7294.2 468.0 637.6 747.5
649.4 738.6

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z, amu
S. Barnes 1/15/10
 Types of fragmentation (2)
IRMPD
• InfraRed Multi-Photon Dissociation
–UUsed d iin FT-ICR
FT ICR iinstruments
t t where
h a vacuum
better than 1 x 10-10 torr is necessary for the
analysis
y of p
peptide
p ions
– The infra-red radiation is delivered by an IR
laser operating at 10.6 microns
– No gas is involved
– In this case, the fragmentation is induced in
the ICR cell
– Effects are essentially equivalent to CID

S. Barnes & M. Renfrow 1/14/11

 ECD and IRMPD with the Finnigan LTQ FT
FTICR MS
• Electron Capture Dissociation
(ECD)
• Infra-Red Multiphoton Dissociation
(IRMPD)
Linear Ion Trap MS
• MS, MS/MS and MSn Analysis by
FTMS
Collision Induced Dissociaiton

Linear Ion Trap 7 T Actively Shielded

Superconducting Magnet

ECD Assembly

IRMPD Laser Assembly

34
 GalNAc

FT-ICR-MS
Gal
Gal

1400 1450 1500 1550 1600 1650 m/z

quadrupole
+ SWIFT
isolated

1400 1450 1500 1550 1600 1650 m/z

GalNAc Gal GalNAc
IRMPD
Gal GalNAc Gal MS / MS

35
1400 1450 1500 1550 1600 1650 m/z
 Types of fragmentation (3)
ECD
• Electron Capture Dissociation
– Used in an ICR cell of an FT
FT-MS
MS instrument
– Low energy electrons interact with the multiply
charged peptide and are absorbed
– They disturb bonding of the peptide backbone
and cleave it without altering the side chain
– Yields c-
c and z-ions
z ions
– MS-MS spectra often very clean, but low
sensitivity
– In conjunction with an IR laser, ECD can
fragment whole proteins (top-down)

S. Barnes & M. Renfrow 1/14/11

 N E D E G pS S S E A D E M A K A L E A E L N D L M

[M + HPO3 + 3H]3+
[M+ HPO3 + 3H]2+• &
+ 80 Da [[M+ HPO3 +2H]]2+
~ x 10

c11 ~x2

c22 2+ z13• ECD

c13 10 ms
c222+
2
z14•
c8 c14
c9 c16
c4 c6 c7
c5 c10 c15 z17• c18
c18 z22•

400 600 800 1000 1200 1400 1600 1800 2000 2200 m/z
Sequencing O-GlcNAc peptides by ECD FT-ICR-MS
Casein kinase II - AGGSTPVSSANMMSG
[Abs. Int. * 1000000]
c V S S A N M

1.5
[M+2H]2+
1.4

1.3

1.2
c8 fragment with sugar attached
1.1

1.0

0.9

0.8

0.7
0.6
990.47346
990 47346
0.5 903.44322 c 9
613.33054 c8 1061.51275 1175.55852 1306.59534
c7 c 10 c 11 c 12
0.4 514.26180
c6
0.3

0.2

0.1

400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z

R1 O R2 O R3

H3 N C C N C C N C COOH
H H H H H

b ion
i cleavage
l c ion
i cleavage
l

S. Barnes & M. Renfrow 1/14/11

CID spectra of Arg-rich peptide

Furry spectra
F t due
d
to -NH3 losses from
Arg residues

Spectra are
uninterpretable

S. Barnes 1/15/10
S. Barnes & M. Renfrow 1/14/11
ECD spectra
p of cystin
y peptide
p p

S. Barnes & M. Renfrow 1/14/11

ECD spectra of phosphorylated
cystin
ti peptide
tid

S. Barnes & M. Renfrow 1/14/11

 IRMPD Fragmentation
F t ti pattern
tt

= GlcNAc
= Fuc
= Man
M
= Xyl

S K PAQ GYGYLG I F N N S K
K. Håkansson, H. J. Cooper, M. R. Emmett, C. E. Costello, A. G. Marshall,
C. L. Nilsson, Anal. Chem. 73, 4530 (2001).
 ECD Fragmentation Pattern

= GlcNAc
= Fuc
= Man
= Xyl

2+ 2+
c4 c5 c6 c7 c8 c9 c10 c15 c16

S K PAQ GYGYLG I F N N S K
z16 z3
2+·

K. Håkansson, H. J. Cooper, M. R. Emmett, C. E. Costello, A. G. Marshall,

C. L. Nilsson, Anal. Chem. 73, 4530 (2001).
 Types of fragmentation (4)
ETD
• Electron Transfer Dissociation
– The electron is provided by an electron
donating chemical species, a radical anion
(azobenzene fluoranthene) directly infused as
(azobenzene,
a reagent gas, or from their precursors
introduced by ESI - 99-anthracenecarboxylic
anthracenecarboxylic
acid, 2-fluoro-5-iodobenzoic acid, and 2-
(fluoranthene-8-carbonyl)benzoic acid)

S. Barnes & M. Renfrow 1/14/11

Electron transfer dissociation

S. Barnes & M. Renfrow 1/14/11

S. Barnes 1/15/10
Syka et al.,
Syka, al PNAS 2004
CAD versus ETD for phosphopeptide

Phosphopeptide loses
phosphate but no sequence
phosphate,
information is obtained

Rich series of c and z ions

S. Barnes & M. Renfrow 1/14/11

ETD better for sulfonated peptides

ETD

S. Barnes & M. Renfrow 1/14/11

Takahashi et al., MCP, 2010
Takahashi et al., MCP, 2010
What affects fragmentation?
g
Chemical composition
p
Peptides
Amino Acid Sequence
Adjacent Amino Acids
Charge State
Location of Charge
Size
Mechanism of Fragmentation
g
Double Bonds
Electrophiles
P t Mobility
Proton M bilit
Gas phase chemistry

S. Barnes 1/15/10
 What can y
you do with a
fragment besides identify?
•Localize PTMs
•Use it to establish signatures for specific ion species
•Biomarker
•Use it to quantify
•Use it to convict a felon
•Add a fragment to create an isobaric tag for quantitative
comparison.
Use it to filter out the information (spectra) you want
•Use
 Tandem mass spectrometry on a
triple
p q quadrupole
p instrument
N2
Gas Collision gas
Sample --+ -+--
-
- -
-
solution
5 KV Q1 Q2 Q3 Detector

• Daughter ion spectra

• Parent ion spectra
• Multiple reaction ion
scanning

S. Barnes & M. Renfrow 1/14/11

N-glycan on aminopeptidase 1

Stephens et al., Eur J. Biochem, 2004

fragmentation pattern of dephosphorylated
lipid A derived from LPS1 (LA1

Moran et al., JBC 2002

Other amino acid fragment ions

S. Barnes & M. Renfrow 1/14/11

Wysocki et al. 2005