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![Page 1: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/1.jpg)
De Novo Sequencing of MS Spectra
Only a manually confirmed spectrum is a correct spectrum
Beatrix UeberheideFebruary 25th 2014
![Page 2: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/2.jpg)
Biological Mass Spectrometry
Time (min)500 1000 1500m/z
Protein(s)
Proteolytic digestion
Peptides
200 600 1000m/z
MS/MSMass Spectrometer
Database Search
Manual Interpretation
MSBase Peak Chromatogram
![Page 3: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/3.jpg)
Peptide Sequencing using Mass Spectrometry
KLEDEELFGS
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
![Page 4: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/4.jpg)
Peptide Sequencing using Mass Spectrometry
K1166
L1020
E907
D778
E663
E534
L405
F292
G145
S88 b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
![Page 5: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/5.jpg)
Peptide Sequencing using Mass Spectrometry
147KL
260E
389D
504E
633E
762L
875F
1022G
1080S
1166 y ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
![Page 6: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/6.jpg)
Peptide Sequencing using Mass Spectrometry
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
[M+2H]2+
762
260 389 504
633
875
292405 534
907 1020663 778 1080
1022
![Page 7: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/7.jpg)
Peptide Sequencing using Mass Spectrometry
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
[M+2H]2+
762
260 389 504
633
875
292405 534
907 1020663 778 1080
1022
113
113
![Page 8: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/8.jpg)
Peptide Sequencing using Mass Spectrometry
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
[M+2H]2+
762
260 389 504
633
875
292405 534
907 1020663 778 1080
1022
129
129
![Page 9: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/9.jpg)
Peptide Sequencing using Mass Spectrometry
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
[M+2H]2+
762
260 389 504
633
875
292405 534
907 1020663 778 1080
1022
![Page 10: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/10.jpg)
Peptide Sequencing using Mass Spectrometry
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
[M+2H]2+
762
260 389 504
633
875
292405 534
907 1020663 778 1080
1022
![Page 11: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/11.jpg)
Peptide Sequencing using Mass Spectrometry
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
m/z
% R
elat
ive
Abu
ndan
ce
100
0250 500 750 1000
[M+2H]2+
762
260 389 504
633
875
292405 534
907 1020663 778 1080
1022
![Page 12: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/12.jpg)
How to Sequence: CAD
Residue Mass (RM)
b ion
b1 = RM + 1
The very first N- and C-terminal fragment ions are not just their corresponding residue masses. The peptides N or C-terminus has to be taken into account.
y ion
y1 = RM + 19
![Page 13: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/13.jpg)
Example of how to calculate theoretical fragment ions
S A M P L E R88 159 290 387 500 629 803
175304417514645716803
Residue MassThe first y ionThe first b ion
![Page 14: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/14.jpg)
How to calculate theoretical fragment ions
S A M P L E R88 159 290 387 500 629 803
175304417514645716803
Residue MassThe first b ion
RM+1 +RM+18
The first y ion
RM+19+ RM + RM + RM + RM + RM+ RM
+ RM + RM + RM+ RM + RM
![Page 15: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/15.jpg)
Finding ‘pairs’ and ‘biggest’ ions
If trypsin was used for digestion, one can assume that the peptide terminates in K or R. Therefore the biggest observable b ion should be:Mass of peptide [M+H] +1 -128 (K) -18Mass of peptide [M+H] +1 -156 (K) -18
y ions are truncated peptides. Therefore subtract a residue mass from the parent ion [M+H] +1 . The highest possible ion could be at [M+H] +1 -57 (G) and the lowest possible ion at [M+H] +1 -186 (W)
b and y ion pairs:Complementary b and y ions should add up and result in the mass of the intact peptide, except since both b and y ion carry 1H+ the peptide mass will be by 1H+ too high therefore: b (m/z) + y (m/z)-1 = [M+H] +1
Check the SAMPLER example
![Page 16: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/16.jpg)
How to start sequencing• Know the charge of the peptide• Know the sample treatment (i.e. alkylation, other
derivatizations that could change the mass of amino acids)
• Know what enzyme was used for digestion• Calculate the [M+1H]+1 charge state of the peptide• Find and exclude non sequence type ions (i.e.
unreacted precursor, neutral loss from the parent ion, neutral loss from fragment ions
• Try to see if you can find the biggest y or b ion in the spectrum. Note, if you used trypsin your C-terminal ion should end in lysine or arginine
• Try to find sequence ions by finding b/y pairs• You usually can conclude you found the correct
sequence if you can explain the major ions in a spectrum
![Page 17: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/17.jpg)
Common observed neutral losses and mass additions:
• Ammonia -17
• Water -18
• Carbon Monoxide from b ions -28
• Phosphoric acid from phosphorylated serine and threonine -98
• Carbamidomethyl modification on cysteines upon alkylation with iodoacetamide +57
• Oxidation of methionine +18Calculate with nominal mass during sequencing, but use the
monoisotopic masses to check if the parent mass fits. For high res. MS/MS check that the residue mass difference is correct.
![Page 18: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/18.jpg)
![Page 19: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/19.jpg)
![Page 20: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/20.jpg)
Mixed Phospho spectra
unmodified
1 Phospho site
1 Phospho site
![Page 21: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/21.jpg)
First ‘on your own example’
Remember what you need to know first!
![Page 22: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/22.jpg)
What is the charge state?
• Neutral loss of water?• Any ions about (z * parent
mass)?• Confirm with b/y pairs!
Neutral loss of water
Water = 18; 18/z; 9
![Page 23: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/23.jpg)
Search for ‘biggest ion’
1433-18-RM1433-18- a residue after which an enzyme cleaves
1433-18-156 = 12491433-18-128 = 1297
![Page 24: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/24.jpg)
1433K
147
1297
![Page 25: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/25.jpg)
1433K
147
1297
87
1210
S
2341433
![Page 26: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/26.jpg)
1433K
147
1297
87
1210
S
2341433
Find the biggest y ion!
Peptide Mass – RMLowest possible ion = GlycineHighest possible ion = Tryptophan
Glycine = 1443-57 = 1386Tryptophan = 1443-186 = 1257
1443-163 = 1280
Y
1280
164
![Page 27: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/27.jpg)
And the sequence is……..
![Page 28: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/28.jpg)
![Page 29: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/29.jpg)
What is the difference ?
Less b ions
A bit of precursor is leftAccurate mass
![Page 30: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/30.jpg)
What if we do not get good fragmentation?
![Page 31: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/31.jpg)
Try a different mode of dissociation
![Page 32: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/32.jpg)
ETD
![Page 33: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/33.jpg)
Electron Transfer Dissociation
CH NHOH
O
O
NH2
NH3+
NH3+
NHR
O
H
+
z' cc''Fluoranthene
+ peptideCH NHOH
O
O
NH2
NH3+
NH3+
NHR
O
H
+
z' cc''Fluoranthene
+ peptideCH NHOH
O
O
NH2
NH3+
NH3+
NHR
O
H
+
z' cc''Fluoranthene
+ peptideCH NHOH
O
O
NH2
NH3+
NH3+
NHR
O
H
+
z' cc''Fluoranthene
+ peptide+
![Page 34: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/34.jpg)
Tandem MS - Dissociation Techniques
CAD: Collision Activated Dissociation (b, y ions)
ETD: Electron Transfer Dissociation (c, z ions)
increase of internal energy through collisions
bombardment of peptides with electrons (radical driven fragmentation)
![Page 35: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/35.jpg)
HPLC LTQ front
Modified rear / CI source
The Prototype Instrument
![Page 36: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/36.jpg)
+
IonDetector
1 0f 2
Three Section RF Linear Quadrupole Trap
Cations
FromESI
Source
3 mTorr HeNICI
Source
Filament
Methane
- Anions
e-
Modifications For Ion/Ion Experiments
Anion Precursor(Fluoranthene)
Secondary RF Supply0-150 Vpeak @ 600 kHz
~700 mTorr
![Page 37: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/37.jpg)
Injection of Positive Ions (ESI)
FrontSection
CenterSection
BackSection
BackLens
FrontLens
++
Ions accumulate
0 V
-10 V
PeptideCations
+++++
++
![Page 38: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/38.jpg)
Precursor Storage in Front Section
+
FrontSection
CenterSection
BackSection
BackLens
FrontLens
0 V
-10 V
Precursor ions moved to front section
![Page 39: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/39.jpg)
Injection of Negative Ions (CI)
Precursor ions held in front section
Negative reagent ionsaccumulate in the center section
+
FrontSection
CenterSection
BackSection
BackLens
FrontLens
0 V
+5 V
---- ----
![Page 40: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/40.jpg)
Charge-Sign Independent Trapping
Pseudo-potentialcreated by+150 Vp 600 kHzappliedto lenses
+0 V
Positive and negative ions react whiletrapped in axial pseudo-potential
-
![Page 41: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/41.jpg)
Charge sign independent radial confinement
+
0 V
-
Axial Confinement With DC PotentialsTrapping is Charge Sign Dependent
![Page 42: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/42.jpg)
Charge sign independent axial confinement with combined RF Quadrupole and
end lens RF pseudo-potentials
+0 V
-
![Page 43: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/43.jpg)
0 V
Reagent AnionsRemoved Axially
Product Cations Trapped in Center Section For Scan Out
+-12 V- -
End ion/ion reactions prepare for product ion analysis
+ + + +
![Page 44: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/44.jpg)
Electron Transfer - Proton Transfer
200 400 600 800 1000 1200 1400
m/z
0
50
100 +3Precursor
Fragmentation(ETD)
![Page 45: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/45.jpg)
200 400 600 800 1000 1200 1400
m/z
0
50
100 +3Precursor
+1+1
+2+2
+3Precursor
0
50
100
200 400 600 800 1000 1200 1400m/z
O -
O
Fragmentation(ETD)
Charge Reduction (PTR)
Electron Transfer - Proton Transfer
![Page 46: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/46.jpg)
The two types of ion reactions
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[M + 3H]2+•
[M + 3H]2+•
Intact Charge-Reduced
Products
Mass ?m/z ?
Charge (z)Sequence ?
Temperature ?Anion ?
He Pressure? FragmentationProducts
c, z, etc.
ET or ETD
![Page 48: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/48.jpg)
[M + 3H]2+•
[M + 3H]2+•
Intact Charge-Reduced
Products
Mass ?m/z ?
Charge (z)Sequence ?
Temperature ?Anion ?
He Pressure? FragmentationProducts
c, z, etc.
ET or ETD
CAD
![Page 49: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/49.jpg)
Charge dependence in fragmentation
![Page 50: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/50.jpg)
Gentle off resonance activation
![Page 51: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/51.jpg)
How to Sequence ETD
Residue Mass (RM)
c1 = RM + 18 z1 = RM + 3
z1 ionc1 ion
+ NH
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Example of how to calculate theoretical fragment ions
S A M P L E R105 176 307 404 517 646 803
159288401498629700803
Residue MassThe first z ion
The first c ion
![Page 53: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/53.jpg)
Largest c and z ions
![Page 54: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/54.jpg)
Sample: Antigens from MHC molecules:
Proline in the 2nd position!
![Page 55: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/55.jpg)
1. What charge state ?[M + H]+1 = m/z (m = m + H)Number of Hs = z
Remember to calculate with nominal masses
![Page 56: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/56.jpg)
(389.5 · 3) – 2 = 1166
(m · z) – (z-1) = [M + H]+1
![Page 57: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/57.jpg)
[M + 2H]+2 = (1166 + 1) /2 = 584
[M + H]+1 + 1H /2
(389.5 · 3) – 2 = 1166
(m · z) – (z-1) = [M + H]+1
check
![Page 58: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/58.jpg)
[M + 2H]+2
[M + 3H]+3·
+3 +2
+1
![Page 59: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/59.jpg)
3. biggest c ion ?
4. biggest z ion ?
2. Eliminate non-sequencing relevant ions!
![Page 60: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/60.jpg)
cbiggest
P I/L1166
1166
1052
130
No suitable zbiggest ion! Remember 2nd position
is a proline?
![Page 61: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/61.jpg)
P I/L1166
1166
1052
130
Now find the first c ion (c1), look for c and z pairs, and sequence ladders
Let’s find c and z pairs!z + c = [M + H]+1 +2
[M + H]+1 – c + 2 = z
![Page 62: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/62.jpg)
P I/L1166
1166
965
116
174 271 358 445 573 701 802
366467595723810897994R S S Q/K SQ/K T I/L
1052
203
![Page 63: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/63.jpg)
2nd Example
![Page 64: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/64.jpg)
Important to know:This sample was converted to Methylesters
(+14 on C-term and D/E side chains) and analyzed after IMAC!
Sample: Antigens from MHC molecules: 9mer
with Proline in the 2nd position!
![Page 65: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/65.jpg)
1. What charge state ?
(372.5 · 3) – 2 = 1115
[M + 2H]+2 = (1115 + 1) /2
[M + H]+1 + 1H /2
= 558
= [M + 2H]+2
check
![Page 66: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/66.jpg)
[M + 2H]+2
[M + 3H]+3·
+3
+2
+1
![Page 67: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/67.jpg)
3. biggest c ion ?
4. biggest z ion ?
2. Eliminate non-sequencing relevant ions!
For c-ions remember to also subtract 14!
![Page 68: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/68.jpg)
c8
No suitable z8 ion! Remember 2nd position
is a proline?
![Page 69: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/69.jpg)
c8
Let’s find c and z pairs!z + c = [M + H]+1 +2
[M + H]+1 – c + 2 = z
![Page 70: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/70.jpg)
c8
Let’s find c and z pairs!z + c = [M + H]+1 +2
[M + H]+1 – c + 2 = z
130201
+2 +2
![Page 71: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/71.jpg)
P I/L1115
1115
987
130201
916A
760
357R
243146Q/K
971 874
399R
718
![Page 72: De Novo Sequencing of MS Spectra Only a manually confirmed spectrum is a correct spectrum Beatrix Ueberheide February 25 th 2014.](https://reader038.fdocuments.in/reader038/viewer/2022103123/56649d895503460f94a6e6b5/html5/thumbnails/72.jpg)
P I/L1115
1115
987
130201
916A
760
357R
243146Q/K
971 874
399R
718pS P P
551 454
566 663