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Protein sequencing and Mass SpectrometryPowerPoint Presentation

Protein sequencing and Mass Spectrometry

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The peptide backbone

The peptide backbone breaks to form

fragments with characteristic masses.

H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH

Ri-1

Ri

Ri+1

C-terminus

N-terminus

AA residuei-1

AA residuei+1

AA residuei

Ionization

The peptide backbone breaks to form

fragments with characteristic masses.

H+

H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH

Ri-1

Ri

Ri+1

C-terminus

N-terminus

AA residuei-1

AA residuei+1

AA residuei

Ionized parent peptide

Fragment ion generation

The peptide backbone breaks to form

fragments with characteristic masses.

H+

H...-HN-CH-CONH-CH-CO-NH-CH-CO-…OH

Ri-1

Ri

Ri+1

C-terminus

N-terminus

AA residuei-1

AA residuei

AA residuei+1

Ionized peptide fragment

Tandem MS for Peptide ID

88

145

292

405

534

663

778

907

1020

1166

b ions

S

G

F

L

E

E

D

E

L

K

1166

1080

1022

875

762

633

504

389

260

147

y ions

100

% Intensity

[M+2H]2+

0

250

500

750

1000

m/z

Peak Assignment

88

145

292

405

534

663

778

907

1020

1166

b ions

S

G

F

L

E

E

D

E

L

K

1166

1080

1022

875

762

633

504

389

260

147

y ions

y6

100

Peak assignment implies

Sequence (Residue tag)

Reconstruction!

y7

% Intensity

[M+2H]2+

y5

b3

b4

y2

y3

b5

y4

y8

b8

b9

b6

b7

y9

0

250

500

750

1000

m/z

Database Searching for peptide ID

- For every peptide from a database
- Generate a hypothetical spectrum
- Compute a correlation between observed and experimental spectra
- Choose the best

- Database searching is very powerful and is the de facto standard for MS.
- Sequest, Mascot, and many others

Spectra: the real story

- Noise Peaks
- Ions, not prefixes & suffixes
- Mass to charge ratio, and not mass
- Multiply charged ions

- Isotope patterns, not single peaks

xn-i

yn-i

yn-i-1

vn-i

wn-i

zn-i

-HN-CH-CO-NH-CH-CO-NH-

CH-R’

Ri

i+1

ai

R”

i+1

bi

bi+1

ci

di+1

low energy fragments

high energy fragments

Peptide fragmentation possibilities(ion types)Ion types, and offsets

- P = prefix residue mass
- S = Suffix residue mass
- b-ions = P+1
- y-ions = S+19
- a-ions = P-27

Mass-Charge ratio

- The X-axis is (M+Z)/Z
- Z=1 implies that peak is at M+1
- Z=2 implies that peak is at (M+2)/2
- M=1000, Z=2, peak position is at 501

- Suppose you see a peak at 501. Is the mass 500, or is it 1000?

Spectral Graph

- Each prefix residue mass (PRM) corresponds to a node.
- Two nodes are connected by an edge if the mass difference is a residue mass.
- A path in the graph is a de novo interpretation of the spectrum

87

G

144

273

332

401

87

144

146

275

100

200

300

S

G

E

K

Spectral Graph- Each peak, when assigned to a prefix/suffix ion type generates a unique prefix residue mass.
- Spectral graph:
- Each node u defines a putative prefix residue M(u).
- (u,v) in E if M(v)-M(u) is the residue mass of an a.a. (tag) or 0.
- Paths in the spectral graph correspond to a interpretation

273

332

401

87

144

146

275

100

200

300

S

G

E

K

Re-defining de novo interpretation- Find a subset of nodes in spectral graph s.t.
- 0, M are included
- Each peak contributes at most one node (interpretation)(*)
- Each adjacent pair (when sorted by mass) is connected by an edge (valid residue mass)
- An appropriate objective function (ex: the number of peaks interpreted) is maximized

87

G

144

273

332

401

87

144

146

275

100

200

300

S

G

E

K

Two problems- Too many nodes.
- Only a small fraction are correspond to b/y ions (leading to true PRMs) (learning problem)
- Even if the b/y ions were correctly predicted, each peak generates multiple possibilities, only one of which is correct. We need to find a path that uses each peak only once (algorithmic problem).
- In general, the forbidden pairs problem is NP-hard

However,..

- The b,y ions have a special non-interleaving property
- Consider pairs (b1,y1), (b2,y2)
- If (b1 < b2), then y1 > y2

0

400

200

Non-Intersecting Forbidden pairs332

300

87

S

- If we consider only b,y ions, ‘forbidden’ node pairs are non-intersecting,
- The de novo problem can be solved efficiently using a dynamic programming technique.

G

E

K

The forbidden pairs method

- There may be many paths that avoid forbidden pairs.
- We choose a path that maximizes an objective function,
- EX: the number of peaks interpreted

100

300

0

400

200

87

The forbidden pairs method- Sort the PRMs according to increasing mass values.
- For each node u, f(u) represents the forbidden pair
- Let m(u) denote the mass value of the PRM.

f(u)

u

D.P. for forbidden pairs

- Consider all pairs u,v
- m[u] <= M/2, m[v] >M/2

- Define S(u,v) as the best score of a forbidden pair path from 0->u, v->M
- Is it sufficient to compute S(u,v) for all u,v?

332

100

300

0

400

200

87

u

v

D.P. for forbidden pairs

- Note that we have one of two cases.
- Either u < f(v) (and f(u) > v)
- Or, u > f(v) (and f(u) < v)

- Case 1.
- Extend u, do not touch f(v)

100

300

0

f(u)

400

200

u

v

The complete algorithm

for all u /*increasing mass values from 0 to M/2 */

for all v /*decreasing mass values from M to M/2 */

if (u > f[v])

else if (u < f[v])

If (u,v)E

/*maxI is the score of the best interpretation*/

maxI = max {maxI,S[u,v]}

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