Transcript Slide 1

Validating sequence
assignments for peptide
fragmentation patterns
by Karen Jonscher
www.proteomesoftware.com
503-244-6027
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With the advent of high throughput proteomics, data is
being generated at an astonishing rate.
It has become clear that validating peptide sequence
assignments generated by database search engines is an
increasingly important, but often overlooked aspect of protein
identification using tandem mass spectrometry.
In this tutorial you will learn about some of the factors
important in low energy peptide fragmentation and how to
use this information to accept or reject database search
engine peptide sequence assignments.
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Why is validating data important?
These two fragment ion spectra from a multi-dimensional LC proteomics
experiment using a quadrupole ion trap were searched with Mascot. They
Every
search,
forequivalent?
a variety of
both had
similar database
scores. Are these
two spectra
•
reasons, generates false positive Mascot
and false
Score = 101
negative assignments. We would like
atspectrum
High to
quality
good signal-toleast reduce, if not eliminate, these with
incorrect
noise and all of the
dominant ions
hits.
assigned.
• Decisions, often involving a great deal of
money for bioassays, will be made
Mascot Score = 102
downstream of our peptide identification.
Inspectrum
Poor quality
with lowthe
signal-tothis era of tight funding, it is crucial that
noise. Fragment ions
are mostrest
likely
data upon which these crucial decisions
randomly assigned to
are completely accurate.
noise peaks.
J
L
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How do peptides break apart?
In order to assess whether a sequence assignment is correct or not,
it is important to understand how and why peptides break apart.
Under low energy dissociation conditions, peptides primarily
fragment at the C – N bond.
If the charge is retained on the N-terminal end of the peptide, the
ion is known as a b-type ion.
If the charge is retained on the C-terminal end, the ion is termed a
y-type ion.
The fragmentation energy in some instruments, especially triple
quadrupoles or quadrupole-time-of-flight hybrids (Q-TOFs) is often
sufficient to generate cleavage at the C-C bond as well, causing
loss of CO from the b ion. These ions are known as a-type ions.
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Peptides dissociate into
nested sets of fragments
H2N -
b1
b2
b3
b4
A
B
C
D
y6
y5
y4
b5
E
y3
b6
F
y2
G - COOH
y1
H2N-A+ =IfbA,
y1 = +G-COOH
1 B, C, D, E, F and G represent
+FG-COOH
H2N-AB+different
= b2
y
=
2
amino acids, this peptide
+
H2N-ABC = b3
y3 = +EFG-COOH
can
dissociate to form the following
+
H2N-ABCD = b4
y4 = +DEFG-COOH
fragments:
+ = b
H2N-ABCDE
y5 = +CDEFG-COOH
5
H2N-ABCDEF+ = b6
y6 = +BCDEFG-COOH
H2N-ABCDEFG+ = b7
y7 = +ABCDEFG-COOH
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Mass difference reflects
peptide sequence
H2N -
b1
b2
b3
b4
A
B
C
D
y6
y5
y4
b5
E
y3
b6
F
y2
G - COOH
y1
Relative Abundance
The peptide amino acid sequence can be deduced by calculating the difference in
mass between peaks. If the mass corresponds to an amino acid residue (a table is
shown in the next slide) then that amino acid is assigned to the peak representing the
FG
E
D
C
B A
difference.
The largest y-type ion will appear
186 amu below the mass
y2 anywhere between
y4 57 to y
5
b2
y
3
of the precursor ion. The smallest y b
ion will appear at the amino acid
y6residue mass
3
b4
b5
plus 19 amu. The largest b ion will be at 18 amu plus the residueb mass below the
6
precursor and the smallest
b ion
AB
C will be at the
D residue
E mass +F1.
G
Using this information, we can interpret fragmentation spectra to deduce the amino
m/z
acid sequence. Keep clicking to view an ion series.
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basic
Name
Symbol
Mass (-H2O)
Side Chain
Immonium/Related Ions
Alanine
A, Ala
71.079
CH3-
44
Arginine
R, Arg
lose ammonia156.188
HN=C(NH2)-NH-(CH2)3-
129/59, 70. 73, 87, 100, 112
H2N-CO-CH2-
87/70
HOOC-CH2-
88/70
HS-CH2-
76
H2N-CO-(CH2)2-
101
Asparagine
acidic
Aspartic acid
-H2S=34
Cysteine
Glutamine
acidic
Glutamic acid
Glycine
basic
Histidine
Isoleucine
Leucine
basic
Lysine
Not
observed
lose ammonia
N, Asnusually
114.104
inD, ion
traps due to
low mass
Asp
115.089
cutoff. Same with b1/y1.
C, Cys
103.145
Q, Gln
lose ammonia128.131
The table of common amino acids
suppress b
provides molecular weights for the
residues, structures of the side chains,
isobaric
and masses for the low mass immonium
isobaric
ions thatlose
result
from side chain loss.
ammonia
-CH3SH=48
Methionine
E, Glu
129.116
HOOC-(CH2)2-
pro102/56, 84, 129
G, Gly
57.052
H-
30
H, His
137.141
N=CH-NH-CH=C-CH2|__________|
110/82, 121, 123, 138, 166
I, Ile
113.160
CH3-CH2-CH(CH3)-
86/44, 72
L, Leu
113.160
(CH3)2-CH-CH2-
86/44, 72
K, Lys
128.17
H2N-(CH2)4-
101/70, 84, 112, 129
M, Met
131.199
CH3-S-(CH2)2-
104/61
These amino acids have chemical
suppress
b
typically
dominant
properties
that
need to be considered
when validating sequence assignments.
Phenylalanine
F, Phe
147.177
Phenyl-CH2-
120/91
Proline
P, Pro
97.117
-N-(CH2)3-CH- |_________|
70
S, Ser
87.078
HO-CH2-
60
lose waterThreonine
T, Thr
101.105
CH3-CH(OH)-
74
Tryptophan
W, Trp
186.213
Phenyl-NH-CH=C-CH2|___________|
159/77, 117, 130, 123, 170, 171
Tyrosine
Y, Tyr
163.176
4-OH-Phenyl-CH2-
136/91, 107
Valine
V, Val
99.133
CH3-CH(CH2)-
72/41, 55, 69
lose water
Serine
abundant y
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Other things to keep in mind
 Basic amino acids can generate
doubly-charged ions.
 Ion signal can be intense for
cleavages C-terminal to acidic amino
acids. These residues also tend to
lose water and cyclize to randomly
eject portions of the sequence.
 Isobaric amino acids cannot be
differentiated using low energy
fragmentation instruments.
 Loss of water from threonine is
particularly intense if the amino acid is
near a terminal end of the peptide.
 If a peptide is tryptic, y1 will either
be lysine at 147 or arginine at 175.
Some pairs of amino acids add up
to the mass of a different amino
acid. The same can happen with
acetylated amino acids, a common
modification.
G-G = 114 = N
G-A = 128 = K/Q
V-G = 156 = R
G-E = 186 = W
A-D = 186 = W
S-V = 186 = W
AcG = 99 = V
AcA = 113 = L/I
AcS = 129 = E
AcN = 156 = R
NEXT
Lxx Val Val Phe
K/Q Gly Arg
We’re going to manually interpret an MS/MS spectrum generated by
a quadrupole ion trap.
1219.4
1337.5
1276.5
1238.6
1139.4
1091.4
992.4
The
The
firstdeconvoluted
ion is at 1337.5. mass
1450- of the precursor is 1449.38 (the observed
1337=113=Lxx,
therefore
we assign the
ion was doubly
charged).
largest y ion as either leucine or
isoleucine.
We’ll
start
byion
looking
the dominant
peaks that are below the mass
Next
we look
at the
at 1238.atAssuming
it is
a The
y of
ion,next
we ion
note
1337-1238=99=Val,
so we
is that
at 1276.
1450-18the
precursor
ion.
assign
the next y ion
as Val.we assign the
1276=156=Arg,
therefore
largest b ion as arginine. Since the sample
We’ll
lookwith
for trypsin,
possible
y ions
between 57 and 186 amu below 1450
was
digested
we would
expect
Continuing
in this+ manner,
weypossible
find
(theor(M+H)
ion)
b ions in a window offset by another
lysine
arginine
as theand
first
ion.b
ions at 1276-1219=57=Gly and
18 amu.
1219-1091=128=Lys/Gln and y ions
at 1238-1139=99=Val and 1139992=147=Phe.
NEXT
Lxx Val Val Phe
K/Q Gly Arg
863.4
To verify the high mass y ion
assignments, we look forSince
thethe largest y ion was either leucine or
complimentary low massisoleucine
b ions. and the next y ion in the series
1219.4
360.2
992.4
was valine, we look for the complementary
ion at 113+1+99=213=b2.
Since the data is from an
ion trap, we
The next b ion will result from addition of
will probably not see b1. another valine, therefore we’d expect a
signal at 213+99=312=b3.
1337.5
1276.5
1139.4
4
1238.6
927.3
944.3
975.4
443.3
459.0
213.0
311.8
232.0
ion at 312+147=459.
1091.4
Therefore, we start byWhen
looking
for b2. is added, we find the b
phenylalanine
NEXT
Lxx Val Val Phe
K/Q
Q Gly Arg
To verify the high mass b ion
assignments, we look for the
complimentary low mass y ions.
863.4
Assuming arginine is y1, we look for a signal
resulting from the addition of glycine at
Since the data is from156+1+18+57=232=y
an ion trap, we 2. We add the 19 amu to
The next complementary y ion should be
will probably not see account
y1, however
the group.
the carboxyl
atfor
232+128=360=y
resulting from the
3
1337.5
1276.5
1238.6
927.3
944.3
975.4
443.3
459.0
311.8
1139.4
1091.4
lysine, since we would have expected a
cleavage there.
Therefore, we start by looking for y2.
232.0
213.0
1219.4
360.2
992.4
assignment of arginine makes
sense
addition
of either lysine or glutamine.
the sample was digested using
given that the sample was Since
digested
trypsin, it is unlikely that the amino acid is
using trypsin.
NEXT
Lxx Val Val Phe Glu
Asn Phe Gln Gly Arg
863.4
507.2
We look for the next largest b ion below
1091. our
There
Validating
high mass ion assignments, we expect to find b5
are three choices: resulting from addition of glutamic acid at 459+129=588=b5 and
The next dominant peak should correspond
to a b ion
y
resulting
from addition of phenylalanine, 360+147=507=y4.
so we
look ybelow
944
and note
that992
9444 Since 992The next
largest
ion
will
appear
below
1091-975=116
830=114=Asn.
863=129=Glu,
we assign
the next y ion as glutamic acid.
1091-944=147=Phe
1091-975=116
1091-944=147=Phe
1091-927=164
1337.5
1276.5
1238.6
1139.4
1091.4
992.4
927.3
944.3
975.4
543.3
489.0
b4
b2
443.3
b3
y2
588.0
830.2
y3
Only the signal at 944 corresponds to an amino acid
residue mass, therefore the next b ion is Phe.
1219.4
1091-927=164
NEXT
Glu Asn Phe Gln Gly Arg
750.3
Lxx Val Val Phe Glu Lxx
621.3
863.4
1219.4
1337.5
1276.5
1238.6
1139.4
1091.4
992.4
830.2
701.1
927.3
944.3
975.4
b5
543.3
489.0
b4
b2
443.3
b3
y2
y3
y4
The next largest y ion will appear below 863. Since
863-750=113=Lxx, we assign the next y ion as
leucine/isoleucine.
The next dominant peak should correspond to a b ion
so we look below 830 and note that 830701=129=Glu.
The next y ion will appear below 750. Since 750621=129=Glu, we assign the next y ion as glutamic
acid. However, since this is complementary to the b
ion we just assigned, our sequence is complete.
1091-975=116
1091-944=147=Phe
1091-927=164
NEXT
b
V
V
F
E
L
E
N
F
Q
G
R
y
y5
L
y11
b11
y10
b8
b5
543.3
489.0
b4
b2
443.3
b3
y2
b9
y9
b7
y3
y8
b10
b5
y7
y4
y6
In this example we
have observed a
complete series of
complementary b
and y ions. Purple
bars indicate b ions
while orange bars
are for y ions.
1091-975=116
1091-944=147=Phe
1091-927=164
NEXT
Incorrect Identification
#
Rank/Sp
--- -------1.
1 / 1
2.
2 / 2
3.
3 / 2
4.
4 / 3
5.
5 / 6
6.
6 / 5
7.
7 / 13
8.
8 / 13
9.
9 / 15
10. 10 / 11
11. 11 / 16
12. 12 / 4
Id#
-------0
0
0
0
0
0
0
0
0
0
0
0
(M+H)+
deltCn
XCorr
Sp
Ions
Reference
Peptide
------------- matches
-----------------to ------Sequest
tenatively
each---spectrum
1450.7694 0.0000 4.7567 2559.1 20/22 CRB1_HUMAN R.LVVFELENFQGR.R
12 peptides.
are 2541.5
rated 20/22
with the
Xcorr R.LVVFELEN*FQGR.R
1451.7534 Matches
0.0254 4.6357
CRB1_HUMAN
0.0571 4.4851 2541.5 20/22 CRB1_HUMAN R.LVVFELENFQ*GR.R
value 1451.7534
(Sequest’s
score
criterion).
HereCRB1_HUMAN
is a
1452.7374
0.2804
3.4230
2036.0 18/22
R.LVVFELEN*FQ*GR.R
1451.7569 0.4038 2.8358 1057.7 15/22 TP3B_HUMAN K.LN*M#VKFLQ*VEGR.G
poorly1450.7729
rated match.
Although most of the
0.4619 2.5595 1426.3 17/22 TP3B_HUMAN K.LN*M#VKFLQVEGR.G
1450.6549
840.0they
13/22were
NEUM_HUMAN
dominant
ions 0.4654
were 2.5430
assigned,
not R.TKQ*VEKN*DDDQ*K.I
1449.6709 0.4752 2.4964
840.0 13/22 NEUM_HUMAN R.TKQ*VEKNDDDQ*K.I
1449.6709
817.4
13/22 NEUM_HUMAN
assigned
to b 0.4757
and y 2.4941
ions but
to water
loss and R.TKQ*VEKN*DDDQK.I
1451.7055 0.5042 2.3586
843.6 13/22 ING_HUMAN
K.S]VETIKEDM#NVK.F
other ions
that0.5145
are generally
less14/22
abundant.
1451.8011
2.3093
817.3
GGT5_HUMAN R.VNVYHHLVETLK.F
1451.7494
0.5179
2.2932
1458.5
16/20
DESP_HUMAN
R.LTYEIEDEKRR.R
NEXT
Correct Identification
#
Rank/Sp
--- -------1.
1 / 1
2.
2 / 2
3.
3 / 2
4.
4 / 3
5.
5 / 6
6.
6 / 5
7.
7 / 13
8.
8 / 13
9.
9 / 15
10. 10 / 11
11. 11 / 16
12. 12 / 4
Id#
-------0
0
0
0
0
0
0
0
0
0
0
0
(M+H)+
deltCn
-------- -----1450.7694 0.0000
1451.7534 0.0254
1451.7534 0.0571
1452.7374 0.2804
1451.7569 0.4038
1450.7729 0.4619
1450.6549 0.4654
1449.6709 0.4752
1449.6709 0.4757
1451.7055 0.5042
1451.8011 0.5145
1451.7494 0.5179
XCorr
-----4.7567
4.6357
4.4851
3.4230
2.8358
2.5595
2.5430
2.4964
2.4941
2.3586
2.3093
2.2932
Sp
---2559.1
2541.5
2541.5
2036.0
1057.7
1426.3
840.0
840.0
817.4
843.6
817.3
1458.5
Ions Reference
Peptide
---- --------------20/22 CRB1_HUMAN R.LVVFELENFQGR.R
20/22 CRB1_HUMAN R.LVVFELEN*FQGR.R
20/22 CRB1_HUMAN R.LVVFELENFQ*GR.R
18/22 CRB1_HUMAN R.LVVFELEN*FQ*GR.R
15/22 TP3B_HUMAN K.LN*M#VKFLQ*VEGR.G
17/22 TP3B_HUMAN K.LN*M#VKFLQVEGR.G
13/22 NEUM_HUMAN R.TKQ*VEKN*DDDQ*K.I
13/22 NEUM_HUMAN R.TKQ*VEKNDDDQ*K.I
13/22 NEUM_HUMAN R.TKQ*VEKN*DDDQK.I
13/22 ING_HUMAN
K.S]VETIKEDM#NVK.F
14/22 GGT5_HUMAN R.VNVYHHLVETLK.F
16/20 DESP_HUMAN R.LTYEIEDEKRR.R
When we compare the incorrect with the correct
identification, we see that all of the dominant ions
are assigned to b and y ions, with water losses
accounting for many of the lower abundance peaks.
The rank is 1 and the Xcorr is high in the Sequest
output file for the correct ID.
NEXT
Score vs. Spectral Quality
As we have seen, a high score may be an
indicator that an identification is correct.
Good Spectral Quality
Good Score
However,
this does not hold true
in all
Bad
ID
Bad ID
cases. In the next few examples,
we will
Good
IDsee
Good ID
instances where good scores actually
corresponded to bad identifications, and bad
scores corresponded to good identifications.
Bad Score
? ID
The importance of spectral quality is
demonstrated and the case of questionable
identification
Bad ID is reviewed.
Bad ID
Bad Spectral Quality
NEXT
Example 1
Proteomics Data
Mascot Search
Good Spectral Quality
Bad ID
Bad Score
Good Score
Bad Spectral Quality
NEXT
Great score, nice spectrum
The mass spectrum has a lot of
1. IPI00001661
Mass: 45425 Total score: 111 Peptides matched: 1
fragment ions and has good
Tax_Id=9606 Regulator of chromosome condensation Check to include this hit in error
signal to noise. Oftentimes,
tolerant search or archive report
good quality spectra like this
Query
Observed
Mr(expt)
Mr(calc)
Delta Miss Score Rank
Peptide
provide good search results.
1
950.77
1899.53
1899.00
0.53
0
111
1
The Mascot score is 111. Usually,
scores over 40 or 50 typically
generate correct identifications.
VVQVSAGDSHTAALTDDGR
The score is well beyond
the 95% confidence level
and is well separated from
the other possibilities,
generally a positive
indicator of a correct ID.
NEXT
The masses of the expected sequence ions are summarized in the data table. Masses labeled
in red were observed in the experiment. As we can see, we have a fairly long run of
contiguous sequence for the y ions and a shorter run for the b ions. There is some overlap of
++
0++
#
b++
b*
b*so
b0
Seq.
y
y++
y*
y*++
y0
y0++
#
the
b band y ions,
however,
we have
a bcomplementary
set.
1
100.14
50.57
V
2
199.27
100.14
V
1800.88
900.94
1783.85
892.43
1782.87
891.94
18
3
327.40
164.21
310.37
155.69
Q
1701.75
851.38
1684.72
842.86
1683.73
842.37
17
4
426.54
213.77
409.51
205.26
V
1573.62
787.31
1556.59
778.80
1555.60
778.30
16
5
513.61
257.31
496.58
248.80
495.60
248.30
S
1474.48
737.75
1457.45
729.23
1456.47
728.74
15
6
584.69
292.85
567.66
284.34
566.68
283.84
A
1387.41
694.21
1370.38
685.69
1369.39
685.20
14
7
641.75
321.38
624.72
312.86
623.73
312.37
G
1316.33
658.67
1299.30
650.15
1298.31
649.66
13
8
756.83
378.92
739.80
370.41
738.82
369.91
D
1259.28
630.14
1242.24
621.63
1241.26
621.13
12
9
843.91
422.46
826.88
413.94
825.90
413.45
S
1144.19
572.60
1127.16
564.08
1126.17
563.59
11
10
981.05
491.03
964.02
482.52
963.04
482.02
H
1057.11
529.06
1040.08
520.54
1039.09
520.05
10
11
1082.16
541.58
1065.13
533.07
1064.14
532.58
T
919.97
460.49
902.94
451.97
901.95
451.48
9
12
1153.24
577.12
1136.21
568.61
1135.22
568.12
A
818.86
409.94
801.83
401.42
800.85
400.93
8
13
1224.32
612.66
1207.29
604.15
1206.30
603.65
A
747.78
374.40
730.75
365.88
729.77
365.39
7
14
1337.48
669.24
1320.45
660.73
1319.46
660.23
L
676.70
338.86
659.67
330.34
658.69
329.85
6
15
1438.58
719.79
1421.55
711.28
1420.57
710.79
T
563.55
282.28
546.51
273.76
545.53
273.27
5
16
1553.67
777.34
1536.64
768.82
1535.65
768.33
D
462.44
231.72
445.41
223.21
444.42
222.72
4
17
1668.76
834.88
1651.73
826.37
1650.74
825.88
D
347.35
174.18
330.32
165.66
329.34
165.17
3
18
1725.81
863.41
1708.78
854.89
1707.79
854.40
G
232.26
116.64
215.23
108.12
2
R
175.21
88.11
158.18
79.59
1
19
19
NEXT
Despite the many of the factors which seemed to lead to a correct
identification, it is wrong. Since this is a good quality spectrum, it
would be worth pursuing other interpretation options. For one, the
peptide may be modified and it would be worth re-searching the data
using a database including modifications such as phosphorylation or
glycosylation, among others. Several searches may be required. De
novo searching would also be a possible approach if modification
Can’t
account
for dominant
searches
do not
provide acceptable
results. ions!!!
Incorrect ID
BUT…
NEXT
Example 2
Good Spectral Quality
Bad ID
Bad Score
Good Score
Bad Spectral Quality
NEXT
All dominant ions are unidentified,
the spectrum is of good quality,
with good signal to noise and well-separated fragment ions
Mascot score is below generally
accepted thresholds.
No significant hits to report
Unassigned queries: (no details means no match) Query Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide
1
868.07
1734.13
1730.98
3.15
2
29
1
KGVASTDNTLIARSLGK
NEXT
There are only short runs of contiguous sequence,
there is little complementarity between the b and y ions.
With dominant ions unidentified the assignment is obviously incorrect.
Since the spectrum is of good quality, the next step should be to consider
modifications.
#
b
b++
b*
b*++
1
129.18
65.10
112.15
56.58
K
2
186.23
93.62
169.20
85.11
G
1603.82
802.41
1586.79
793.90
1585.80
793.40
16
3
285.37
143.19
268.34
134.67
V
1546.76
773.89
1529.73
765.37
1528.75
764.88
15
4
356.45
178.73
339.42
170.21
A
1447.63
724.32
1430.60
715.80
1429.62
715.31
14
5
443.52
222.27
426.49
213.75
425.51
213.26
S
1376.55
688.78
1359.52
680.27
1358.54
679.77
13
6
544.63
272.82
527.60
264.30
526.61
263.81
T
1289.48
645.24
1272.44
636.73
1271.46
636.23
12
7
659.72
330.36
642.69
321.85
641.70
321.36
D
1188.37
594.69
1171.34
586.17
1170.35
585.68
11
8
773.82
387.41
756.79
378.90
755.81
378.41
N
1073.28
537.14
1056.25
528.63
1055.27
528.14
10
9
874.93
437.97
857.90
429.45
856.91
428.96
T
959.18
480.09
942.15
471.58
941.16
471.09
9
10
988.09
494.55
971.06
486.03
970.07
485.54
L
858.07
429.54
841.04
421.02
840.06
420.53
8
11
1101.25
551.13
1084.21
542.61
1083.23
542.12
I
744.91
372.96
727.88
364.45
726.90
363.95
7
12
1172.32
586.67
1155.29
578.15
1154.31
577.66
A
631.75
316.38
614.72
307.87
613.74
307.37
6
13
1328.51
664.76
1311.48
656.24
1310.50
655.75
R
560.67
280.84
543.64
272.33
542.66
271.83
5
14
1415.59
708.30
1398.56
699.78
1397.57
699.29
S
404.49
202.75
387.46
194.23
386.47
193.74
4
15
1528.75
764.88
1511.72
756.36
1510.73
755.87
L
317.41
159.21
300.38
150.69
3
16
1585.80
793.40
1568.77
784.89
1567.79
784.40
G
204.25
102.63
187.22
94.11
NEXT 2
K
147.20
74.10
130.17
65.59
1
17
b0
b0++
Seq.
y
y++
y*
y*++
y0
y0++
#
17
Example 3
Good Spectral Quality
Good ID
Bad Score
Good Score
Bad Spectral Quality
NEXT
Score = 31  below threshold
BUT…
All dominant ions accounted for
Largest peak corresponds to y5 – cleavage at proline
#
b
b0
Seq.
1
72.09
A
2
129.14
G
y
y*
y0
#
15
1391.57
14
3
186.19
1352.53 1335.50 1334.52
•Chemistry
isG plausible
4
257.27
A
1295.48 1278.45 1277.46
Sequest
search
provided
Water loss for y ions also starts after•A
5
328.35
A
1224.40 1207.37 1206.38
the threonine.
6
427.48
V
1153.32 1136.29as
1135.31
the
same identification
7
526.61
V
1054.19 1037.16 1036.17
Possibly the presence of the basic the
Mascot search
histidine and the acidic glutamic acid 8 639.77
I
955.06
938.03
937.04
correct
assignment
inhibit the water loss at y and y . •Likely
9
740.88
722.86
T
841.90
824.87
823.88
13
No water loss for b ions until b9
when Thr appears.
3
1409.58
1392.55
12
11
10
4
9
8
7
10
869.99
851.98
E
740.79
723.76
722.78
6
11
967.11
949.10
P
611.68
594.65
593.66
5
12
1096.23
1078.21
E
514.56
497.53
496.54
4
13
1233.37
1215.35
H
385.44
368.41
367.43
3
14
1334.47
1316.46
T
248.30
231.27
230.29
2
K
147.20
130.17
15
1
NEXT
Example 4
Good Spectral Quality
Bad Score
Good Score
Bad ID
Bad Spectral Quality
NEXT
Score = 18 Very weak spectrum
There is no baseline, thus we must assume this
is a noise spectrum. The ions are likely
assigned by random chance.
NEXT
#
b
b++
1
58.06
29.53
2
157.19
79.10
3
228.27
114.64
4
299.35
150.18
5
412.51
206.76
6
513.61
257.31
7
600.69
300.85
8
715.78
358.39
9
812.90
406.95
10
883.98
442.49
11
983.11
492.06
12
1111.24
556.12
13
1182.32
14
Although there are some complementary
runs of contiguous sequence ions, it is
unlikely they are significant, given the
quality of the mass spectrum.
b*
b*++
b0
b0++
Seq.
y
y++
y*
y*++
y0
y0++
G
495.60
248.30
#
25
V
2413.73
1207.37
2396.70
1198.85
2395.71
1198.36
24
A
2314.60
1157.80
2297.57
1149.29
2296.58
1148.79
23
A
2243.52
1122.26
2226.49
1113.75
2225.50
1113.25
22
L
2172.44
1086.72
2155.41
1078.21
2154.42
1077.72
21
T
2059.28
1030.14
2042.25
1021.63
2041.26
1021.14
20
We do see a dominant fragment ion at the
y17 proline, however other large signals
do not correspond to expected cleavages
C-terminal of the acidic amino acids.
582.68
291.84
S
1958.17
979.59
1941.14
971.08
1940.16
970.58
19
697.77
349.39
D
1871.10
936.05
1854.06
927.54
1853.08
927.04
18
794.88
397.95
P
1756.01
878.51
1738.98
869.99
1737.99
869.50
17
865.96
433.48
A
1658.89
829.95
1641.86
821.43
1640.87
820.94
16
965.09
483.05
V
1587.81
794.41
1570.78
785.89
1569.80
785.40
15
1094.21
547.61
1093.23
547.12
Q
1488.68
744.84
1471.65
736.33
1470.66
735.84
14
591.66
1165.29
583.15
1164.30
582.66
A
1360.55
680.78
1343.52
672.26
1342.53
671.77
13
1295.48
648.24
1278.45
639.73
1277.46
639.24
I
1289.47
645.24
1272.44
636.72
1271.45
636.23
12
15
1394.61
697.81
16
1507.77
754.39
17
1622.86
811.93
18
1723.96
862.49
19
1795.04
898.03
20
1882.12
941.56
21
1997.21
22
The assignment of doubly-charged ions
without the presence of a basic ion is
highly unlikely, therefore this identification
is incorrect.
1377.58
689.29
1376.60
688.80
V
1176.31
588.66
1159.28
580.14
1158.29
579.65
11
1490.74
745.87
1489.76
745.38
L
1077.18
539.09
1060.15
530.58
1059.16
530.08
10
1605.83
803.42
1604.84
802.93
D
964.02
482.51
946.99
474.00
946.00
473.51
9
1706.93
853.97
1705.95
853.48
T
848.93
424.97
831.90
416.45
830.91
415.96
8
1778.01
889.51
1777.03
889.02
A
747.82
374.42
730.79
365.90
729.81
365.41
7
1865.09
933.05
1864.11
932.56
S
676.74
338.88
659.71
330.36
658.73
329.87
6
999.11
1980.18
990.59
1979.19
990.10
D
589.67
295.34
572.64
286.82
571.65
286.33
5
2096.34
1048.68
2079.31
1040.16
2078.33
1039.67
V
474.58
237.79
457.55
229.28
456.56
228.79
4
23
2209.50
1105.26
24
2324.59
1162.80
25
This spectrum could likely be discarded.
2192.47
1096.74
2191.49
1096.25
L
375.45
188.23
358.42
179.71
357.43
179.22
3
2307.56
1154.28
2306.58
1153.79
D
262.29
131.65
245.26
123.13
244.27
122.64
2
K
147.20
74.10
130.17
65.59
NEXT
1
Example 5
Good Spectral Quality
Good Score
? ID
Bad Spectral Quality
Bad Score
NEXT
Scorebelow
= 38 threshold, spectral quality OK but
Score slightly
fragmentation is limited, questionable ID
Most abundant peak
results from loss of water
The identification could possibly be disregarded if the protein
from serine, not proline
assignment was confirmed by another peptide.
cleavage as expected.
The doubly-charged y8
ion is reasonable, given
the presence of the
arginine. However, the
b2 doubly-charged ion is
unlikely.
#
b
b++
1
132.20
66.60
2
261.32
131.16
243.30
122.15
E
945.06
473.04
928.03
464.52
927.05
464.03
8
3
358.43
179.72
340.42
170.71
P
815.95
408.48
798.92
399.96
797.93
399.47
7
4
472.54
236.77
455.51
228.26
454.52
227.76
N
718.83
359.92
701.80
351.40
700.82
350.91
6
5
559.61
280.31
542.58
271.80
541.60
271.30
S
604.73
302.87
587.70
294.35
586.71
293.86
5
6
672.77
336.89
655.74
328.38
654.76
327.88
L
517.65
259.33
500.62
250.81
499.63
250.32
4
7
828.96
414.98
811.93
406.47
810.95
405.98
R
404.49
202.75
387.46
194.23
386.47
193.74
3
8
930.07
465.54
913.04
457.02
912.05
456.53
T
248.30
124.66
231.27
116.14
230.29
115.65
2
K
147.20
74.10
130.17
65.59
9
b*
b*++
b0
b0++
Seq.
y
y++
y*
y*++
y0
y0++
#
M
9
NEXT
1
Example 6
Good Spectral Quality
Good ID
Bad Score
Good Score
Bad Spectral Quality
NEXT
Score = 79
Complete b and y series
Plausible ion chemistry
Correct ID!
Abundant ions at D
and W cleavages
#
b
b++
b*
b*++
b0
b0++
Seq.
y
y++
y*
y*++
y0
y0++
1
132.20
66.60
2
233.31
117.16
215.29
108.15
T
1689.83
845.42
1672.79
836.90
1671.81
836.41
13
3
348.39
174.70
330.38
165.69
D
1588.72
794.86
1571.69
786.35
1570.71
785.86
12
4
476.52
238.77
459.49
230.25
458.51
229.76
Q
1473.63
737.32
1456.60
728.80
1455.62
728.31
11
5
605.64
303.32
588.61
294.81
587.63
294.32
E
1345.50
673.25
1328.47
664.74
1327.49
664.25
10
6
676.72
338.86
659.69
330.35
658.70
329.86
A
1216.39
608.70
1199.35
600.18
1198.37
599.69
9
7
789.88
395.44
772.85
386.93
771.86
386.44
I
1145.31
573.16
1128.28
564.64
1127.29
564.15
8
8
918.01
459.51
900.98
450.99
899.99
450.50
Q
1032.15
516.58
1015.12
508.06
1014.13
507.57
7
9
1033.10
517.05
1016.07
508.54
1015.08
508.05
D
904.02
452.51
886.99
444.00
886.00
443.50
6
10
1146.26
573.63
1129.23
565.12
1128.24
564.63
L
788.93
394.97
771.90
386.45
5
11
1332.47
666.74
1315.44
658.22
1314.46
657.73
W
675.77
338.39
658.74
329.87
4
12
1460.60
730.80
1443.57
722.29
1442.59
721.80
Q
489.55
245.28
472.52
236.77
3
13
1646.82
823.91
1629.78
815.40
1628.80
814.90
W
361.42
181.22
344.39
172.70
NEXT2
R
175.21
88.11
158.18
79.59
1
14
M
#
14
The following examples compare spectra
that were acquired on a Q-TOF and an
ion trap.
Both correct and incorrect identifications
are shown.
The different appearance of the
fragmentation spectra, and the presence
of immonium ions and low mass fragment
ions in the Q-TOF, means that different
considerations may need to be taken
into account when different instruments
are being used.
NEXT
Correct Identification
Note the presence of y1 and
the immonium ion for F in
the QTOF spectrum. The
QTOF spectrum shows b
ion suppression except for
b2.
QTOF
Ion Trap
NEXT
Incorrect Identification
Both spectra have many dominant ions
unaccounted for. The dominant ions
are assigned as b type in the QTOF
spectrum, which is unlikely.
QTOF
Ion Trap
NEXT
Look at the next two slides.
Can you guess which are the
correct identifications and
which are incorrect?
NEXT
QTOF
Ion Trap
NEXT
QTOF
Ion Trap
NEXT
Summary
When interpreting the results of search
engines it is important to
– Look at the score
– Look at the sequence runs
– Consider the ion fragmentation chemistry
– What about the instrument?
– Does it all make sense?
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