Proteome Informatics. Brian C. Searle Creative Commons Attribution
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1 Proteome Informatics Brian C. Searle Creative Commons Attribution
2 Section structure Class 1 Class 2 Homework 1 Mass spectrometry and de novo sequencing Database searching and E-value estimation Implementing a simple database search engine Class 3 Mixture model analysis and target/decoy evaluation Class 4 Searching for post-translational modifications Homework 2 Evaluating mixture models
3 de Novo Sequencing Tandem Mass Spectra Taylor JA, Johnson RS. Rapid Commun Mass Spectrom. 1997;11(9): Brian C. Searle Creative Commons Attribution
4 Proteins are digested into peptides with trypsin F V N Q H L C G S K L T I R P T E R C G A E R V L Y L V A E 1. FVNQHLCGSK 2. LVEALYLVCGER 3. AEPTIR 4. TR
5 There are hundreds of thousands of peptides in the human proteome Intensity (10 9 ) M/Z ,000 M/Z Mass/Charge (m/z)
6 Liquid chromatography separates peptides by hydrophobicity intensity intensity intensity intensity m/z m/z m/z m/z
7
8 Sequencing peptides with data dependent acquisition (DDA)
9 DDA relies on dynamic exclusion 400 M/Z 1000 M/Z Retention TIme
10 Sequencing AEPTIR F V N Q H L C G S K L T I R P T E R C G A E R V L Y L V A E 1. FVNQHLCGSK 2. LVEALYLVCGER 3. AEPTIR 4. TR
11 Impart energy in collision cell A E P T I R H 2 O Name AA Mono Glycine G Alanine A Serine S Proline P Valine V Threonine T Cysteine C Isoleucine I Leucine L Asparagine N Name AA Mono Aspartic Acid D Glutamine Q Lysine K Glutamic Acid E Methionine M Histidine H Phenylalanine Y Arginine R Tyrosine Y Tryptophan W
12 Measure mass of daughter ions A E P T A E P Intensity A A 72.0 E M/z
13 B-type Ions A E P T I R H 2 O Intensity M/z
14 B-type Ions A E P T I R H 2 O Intensity A-0 AE-A AEP -AE AEPT -AEP AEPTI -AEPT AEPTIR -AEPTI M/z
15 B-type Ions A E P T I R H 2 O Intensity CO CO CO CO CO CO M/z
16 A-type Ions A E P T I R H 2 O Intensity CO CO CO CO CO CO M/z
17 Y-type Ions H 2 O R I T P E A Intensity M/z
18 Y-type Ions H 2 O R I T P E A Intensity M/z
19 B-type, A-type, Y-type Ions H 2 O R I T P E A Intensity M/z
20 Intensity M/z
21 Intensity M/z
22 Intensity M/z
23 A E P T I R H 2O Intensity M/z
24 Known Ion Types B-type ions A-type ions Y-type ions
25 Known Ion Types B-type ions A-type ions Y-type ions B- or Y-type +2H ions B- or Y-type -NH 3 ions B- or Y-type -H 2 O ions
26 Known Ion Types B-type ions A-type ions Y-type ions 100% 20% 100% B- or Y-type +2H ions B- or Y-type -NH 3 ions B- or Y-type -H 2 O ions 50% 20% 20%
27 Model Spectrum ELVISLIVESK *Courtesy of Dr. Richard Johnson
28 B/Y type ions (100%) B/Y +2H type ions (50%) A type ions B/Y -NH 3 /-H 2 O (20%)
29 Model Spectrum
30 Model Spectrum
31 Model Spectrum
32 How would you sequence this peptide? 132 intensity mass / charge
33 Brute force strategy AAAA AACA NYYY AAAC AACC PYYY AAAD AACD RYYY AAAE AAAF AACE AACF SYYY TYYY AAAG AACG VYYY AAAH AACH WYYY AAAI AACI YYYY
34 Can look for a tag, but how do you know which direction? 132 intensity N 374 A NA or AN? 426 mass / charge
35 Can use ends as context: start at y1/b1 and keep going 132 intensity I/L K/Q mass / charge
36 Shortest path algorithm with a directed acyclic graph (DAG) (B) 1 (Y)
37 Completely interconnected graph (B) 1 (Y)
38 Graph with only amino acid edges (B) 1 N I/L I/L G G A (Y) K/Q [CH] K/Q
39 Convert edges to scores What are good things to score? Amino acid frequency Ion intensities Ion frequencies Likelihood ion produces intense signal
40 Dijkstra s Shortest Path Algorithm END START
41 Travel the smallest known edge (1) END START Update: Michael was right -- you don t need to multiply by -1
42 Travel the smallest known edge (2) END START
43 Travel the smallest known edge (3) END START
44 Travel the smallest known edge (4) END START
45 Travel the smallest known edge (5) END START
46 Graph with only amino acid edges (B) 1 N I/L I/L G G A (Y) K/Q [CH] K/Q
47 DAG with Topographical Support (1)
48 DAG with Topographical Support (2)
49 DAG with Topographical Support (3)
50 DAG with Topographical Support (4)
51 DAG with Topographical Support (5)
52 DAG with Topographical Support (6)
53 DAG with Topographical Support (7)
54 Trace the path backwards through the DAG
55 Sequencing the peptide (B) 1 N (Y) 19 I/L I/L G G A K/Q [CH] K/Q AN[I/L][K/Q]
56 How would you sequence this peptide? 132 intensity mass / charge
57 How would you sequence this peptide? intensity 132 (y1) 147 (y2) (y3) 374 (y4) 426 K/Q I/L N A
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