Technology & Methods Seminar “Blast and Other Methods of Probabilistic Sequence Comparison”...

Technology & Methods Seminar

“Blast and Other Methods of Probabilistic Sequence Comparison”

Arcady MushegianBioinformatics

Thursday, May 25, 1:00p.m.Classroom

(1st floor, Administration Building)

Schedule with abstracts and previous presentation slides can be found on:K:\Weekly Seminar Schedule\Thursday -- Technology & Methods

Information regarding previous seminars can be found at:http://research.stowers-institute.org/wiw/external/Seminars/index.htm

..

Laurence [email protected]

ProteomicsRooms 351-357

Over 2000 Samples Analyzed since Oct 2003

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Protein List

Peptide MixtureCellsTissues

EmbryosDigestion

Proteins

High-Throughput Proteomics: MudPIT

2D Chromatography

Database MatchingMS/MS Spectrum

Data Analysis

Tandem Mass Spectrometry

LysisPurification

Complex Protein MixturesPeptide MixtureCells

Tissues Embryos

Digestion

Proteins

2D Chromatography

MS/MS Spectrum


LysisPurification

Database Matching

Data Analysis

Protein List

Complex Protein Mixtures Types of samples we deal with:

Whole cell lysates Membrane/Organelle preps Co-IPed proteins Affinity purified proteins

Any biochemically-sound protein mixture and appropriate negative controls

What we need to know about your samples: Details of prep:

Organism Amino acid sequence(s) Buffer

Concentration, Silver-stained gel, Western Blot Protein content or PTMs

Dat

abas

e

Chr

omat

oM

ass

Spec

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a An

alys

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Dig

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Drop off: -80°C Freezer (Hallway Room 351)

Quantities: low µg to 500µg

Sample form: Small starting volume (<50ul) can be digested directly Dried protein pellet after TCA-precipitation (PREFERRED)

Complex Protein Mixtures - Troubleshooting

RT: 0.00 - 20.02

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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0 .79

1.801.00

2.01 18.30

2.15

1.09 2.22

2.50

2.5718.262.82 2.96

3.023.43

3.50 6.94 7.236.303.79 6.23 7.828.41

8.7319.9319.219.01

12.62 15.1610.53 14.61 15.96 18.0816.5111.63

NL:1.11E9

Bas e Peak MS ScMcd1-Myc_Meta_Ti_101

RT: 0.00 - 117.08

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97.63 107.9413.19 103.3395.7583.0074.50 112.4210.99 90.38

89.4213.8484.82

14.10

10.40 72.09

71.42

70.43

69.5168.86

68.60

65.99

65.40

64.8247.99

63.32

62.27

54.66 60.639.98

53.57

9.7652.2646.08

9.13 15.38

5.75 40.8240.27

5.26

34.054.57

22.6520.33 32.68

NL:4.29E9


RT: 0.00 - 117.00

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84.10 116.6910.448.25 86.334.15 83.56 108.5299.4789.9480.03

3.38

7.2411.11

97.2279.2577.28 94.19 111.66

76.50

14.11

73.93

71.43

66.6865.36

63.2861.75

60.70

59.2952.75

52.58

52.33

44.36 51.0943.35

38.3837.6915.24 34.21

15.90 20.88 28.54

NL:4.27E9


RT: 0.00 - 117.02

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3 .62 5.41 9.00 12.61

102.65

101.42 107.77114.87

112.4687.31

12.85

98.4895.58

90.0413.58

94.99

86.35

84.46

82.96

81.2676.39

74.89

73.5945.62 72.2470.17

67.5960.85

53.5652.57

53.72

54.1215.7645.16 51.93

23.9718.93 40.0435.5829.18

NL:4.28E9


RT: 0.00 - 117.03

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0 .315.27

5.57 11.083.82

12.25

12.50

12.79

13.43

105.69104.01 110.85

97.55

115.2694.70

89.51

78.79 88.2676.0370.7552.70

67.5814.46 53.5419.57 60.6024.59 29.85 45.1234.98 39.99

NL:3.87E9


RT: 0.00 - 117.03

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101.06 104.8696.94105.10 114.70

112.6594.62

111.38

105.99

92.73

15.50

91.20

90.43

15.70

90.27

89.40

10.19

11.379.6586.73

7.666.5586.38

77.3075.9671.2962.0649.10 83.4926.6523.03 44.19 54.5340.9230.43

NL:4.28E9


Problems with “Sticky Stuff”: Glycerol High Detergent (e.g. 2% SDS) DNA/RNA

Solutions: Keep as low as possible or dialyze out Methanol/Chloroform extraction Benzonase

RT: 0.00 - 116.97

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Tim e (m in)

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62.49

89.81

62.84

39.52

47.81

44.6293.19

56.40

37.6763.14

52.16

15.16

58.1136.88

15.7773.68

103.6183.04

16.02 94.0888.3914.47 81.0065.38

74.5616.33 68.26 99.9526.60 35.7124.5513.89 104.382.67 4.38 109.83

NL:3.07E9

Base Peak MS ScMcd1-Myc_Meta_Ti_402

RT: 0.00 - 117.05

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Tim e (m in)

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54.39

61.15

60.86

61.70

49.1042.2040.5233.81

77.4039.5312.6213.3011.79 47.766.05 58.59 67.15 80.0069.31 116.7944.8220.62 21.88 111.1285.8532.02 107.2999.7187.94

NL:1.63E9


RT: 0.00 - 116.96

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39.72

54.25

60.68

60.85

40.47

45.69

44.5061.33

34.71

48.7320.98 58.3212.77 22.5312.02 67.036.32 32.85 115.4950.28 75.92 78.37 112.1882.45 89.21 106.3293.72

NL:3.23E9


RT: 0.00 - 117.05

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54.86

39.77

61.77

45.12

46.42

43.18

35.97

62.38

31.5662.5646.78

50.8523.0114.0512.51 23.8214.858.17 58.53 115.10106.3226.07 67.93 82.0178.8116.40 102.4588.5275.03 91.16

NL:1.88E9


RT: 0.00 - 97.06

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Tim e (m in)

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60.58

62.41

58.27

92.36

92.03

90.91

90.20

88.97

87.8773.7013.51 81.91 84.4680.1414.0712.37

78.6512.11 14.79

65.7311.20

66.66 73.2410.667.55

6.64 57.814.17

56.7616.1453.91

18.98 47.1126.92 36.1529.17 40.29

NL:1.44E9


RT: 0.00 - 20.05

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3.45

3.962.85

4.02

4.22

2.71

4.29

4.35

2.302.22

4.421.71

1.64

1.51

1.444.49

4.561.38

4.831.311.03 5.07

5.24 18.705.49 14.65 17.4416.9116.14 19.3213.7013.0612.3110.619.639.256.24 7.28 8.64

NL:3.86E9


13 IDed Proteins 67 IDed Proteins

w/o Benzonase w/ Benzonase


EmbryosDigestion

ProteinsLysis

Purification

Generating Peptides

2D Chromatography

MS/MS Spectrum


Database Matching

Data Analysis

Protein List

Generating PeptidesProteomic Surveying: Endoproteinase Lys-C + Trypsin

MKLSEVFEQE IDPVMQSLGY CCGRKLEFSP QTLCCYGKQL CTIPRDATYY SYQNRYHFCE KCFNEIQGES VSLGDDPSQP QTTINKEQFS KRKNDTLDPE LFVECTECGR KMHQICVLHH EIIWPAGFVC DGCLKKSART RKENKFSAKR LPSTRLGTFL DSMCRLELKL NSS

Proteomic Surveying: Endoproteinase Lys-C + Trypsin


PTMs: High Sequence Coverage

MSQAIAEKQP SQEVKMEAKM EVDQPEPADT QPEDISESKV EDCKMESTET EERSTELKTE IKEEEDQPST SATQSSPAPG QSKKKIFKPE ELRQALMPTL EALYRQDPES LPFRQPVDPQ LLGIPDYFDI VKSPMDLSTI KRKLDTGQYQ EPWQYVDDIW LMFNNAWLYN RKTSRVYKYC

Generating Peptides

• Sample is split into x aliquots

• Digest using x different proteases

• Analyze samples individually

• Interpret spectra using SEQUEST

MudPITMudPIT MudPIT

Trypsin Subtilisin Elastase

MacCoss et al. (2002) Proc Natl Acad Sci U S A. 99:7900-7905

Post-Translational Modifications: Triple/Multiple Digest

Generating PeptidesProteomic Surveying: Endoproteinase Lys-C + Trypsin


PTMs: High Sequence Coverage

MSQAIAEKQP SQEVKMEAKM EVDQPEPADT QPEDISESKV EDCKMESTET EERSTELKTE IKEEEDQPST SATQSSPAPG QSKKKIFKPE ELRQALMPTL EALYRQDPES LPFRQPVDPQ LLGIPDYFDI VKSPMDLSTI KRKLDTGQYQ EPWQYVDDIW LMFNNAWLYN RKTSRVYKYC

5 Ti + 2 Es + 1 PK 86.6% SP

Ext

race

llula

rGenerating Peptides: DmSNS (1479 AA, 162kDa)

Maggie Chen, Kiran Kocherlakota, Jeff McDermott …

Cyt

odom

ain

Maggie Chen, Kiran Kocherlakota, Jeff McDermott

Generating Peptides - Troubleshooting

No tryptic peptides are obtained from particular protein Overdigestion with non-specific proteases No positively charged amino acids in peptides


EmbryosDigestion

ProteinsLysis

Purification

Multidimensional Chromatography

MS/MS Spectrum


Database Matching

Data Analysis

2D Chromatography

Protein List

Small digest volumes (<200µl): 3-phase 100µm Fused Silica

SCX

Peptides

RP - C18

RP - C18

100µm FS

Multidimensional Chromatography

Peptides

100µm FS

wasteFiltered Unionwith 2µm Frit

250µm FS

250µm FS

SCX

RP - C18

RP - C18

250µm FS

Large digest volumes (>200µl): split-3-phase 250µm Fused Silica

HPLC

H2O MeCN NH4OAc

0 20 40 60 80 1000

20

40

60

Bu

ffer

(%

)

Time (min)

Multidimensional Chromatography• Cycle 1 – Reverse Phase Gradient

• Cycle 2_Step 1 – Salt Pulse

• Cycle 2_Step 2 – Reverse Phase Gradient

• Cycle 3_Step 1 – Increase Salt Pulse

• Cycle 3_Step 2 – Reverse Phase Gradient

2.4kV

Number of Cycles depends on Sample Complexity


EmbryosDigestion

ProteinsLysis

Purification


Database Matching

Data Analysis

2D Chromatography


MS/MS Spectrum

Protein List


Hardware improvements for LTQ: Trapping efficiency (no rf field in ion injection axis), Ion capacity (linear configuration of mass analyzer -> larger volume), ~2x Detection efficiency (radial ejection of ions + 2 detectors) ~3x Ion ejection rate (while maintaining same resolution) Should significantly improve the number of detected peptides/proteins

Deca-XP vs LTQ(3D vs Linear Ion Trap)

Blackler et al. (2006) Anal. Chem.78:1337-13344

Tandem Mass SpectrometryDeca-XP vs LTQ

~2.5x more Protein IDs More Proteins that matter: not necessarily More spectra per protein (Quantitation, PTMs)

~10µg FLAG-tagged Mediator Prep

Both machines: Detection limit is ~0.05% of Total Protein Quantity (w/w) LTQ: more peptides/spectra detected for same quantities, i.e. greater confidence

LTQVarying amounts ofPfs25 (185AA, 20kDa)Constant HsMediator (~10µg Total)

XPVarying amounts of SpRunt1 (535AA, 59kDa)Constant HsMediator (~20µg Total)



The increase in number of spectra obtained from the LTQ results in: much larger files (x10) significantly increase in computational overhead (Storage and Search Time)

Blackler et al. (2006) Anal. Chem.78:1337-13344

Computational Overhead

Dan Thomasset

Instrumentation Priorities

XP On-going Protein Identification Projects

LTQ1. Quantitation (isotopic labeling)2. PTMs3. Low Abundance Proteins4. New Protein Identification Projects


EmbryosDigestion

ProteinsLysis

Purification

Data Analysis

2D Chromatography


Database MatchingMS/MS Spectrum

Matching MS/MS Spectra to Peptides

Protein List

Matching MS/MS Spectra to Peptides

Protein Sequence Databases: Need to be as comprehensive as possible (whole genomes) Need to include “custom” sequences Updates

Shuffled Sequences: Used to estimate False Discovery Rates (FDR)

ralCountTotalSpect

untSpectralCoShuffledxFDR

)_(2

Search engine: SEQUEST®


EmbryosDigestion

ProteinsLysis

Purification

Data Analysis

2D Chromatography


MS/MS SpectrumDatabase Matching

Protein List

Data Analysis

FILTER

ASSEMBLEPARSE

SQTs

Filtering & Assembling Data: DTASelect

DTASelect

High-stringency Filtering Criteria to limit FDR: Cross-correlation score (XCorr) DeltaCN Peptide Length Peptide Ends

Through P: drive http://bioinfo/proteomics/

DTASelect.html (DTASelect-filter.txt)

Tabb et al. (2002) J Proteome Res 1: 21-26

COMPARE

Comparing Protein Lists: CONTRASTn Protein Lists

MERGE

Tabb et al. (2002) J Proteome Res 1: 21-26

MSAccessMSAccess

Text files

CONTRAST

Contrast.html (Contrast.txt)

COMPARE

Comparing Protein Lists: contrast-reportn Protein Lists

contrast-report

report.xls

Dan He

Mike Coleman

Relative Abundance: NSAF

N

ii

kk

LengthuntSpectralCo

LengthuntSpectralCoNSAF

1

)/(

)/()(

Normalized Spectral Abundance Factor

Quantitative Information from MudPIT dataset? Sequence Coverage: high for small proteins / low for large proteins Spectral Count: large proteins contribute more peptides/spectra

Values between 0 and 1 Best approximation of protein levels in a sample Allows comparisons across multiple runs and across different instruments

Relative Abundance: NSAF

Erika Geisbrecht Mingan Shi

Modification Delta Mass Residues Symbol

Methylation 14 KST #

Hydroxylation 16 KD @

Oxidation 16 M @

Dimethylation 28 RK ~

Acetylation 42 KST ^

Phosphorylation 80 STY *

Ubiquitination 114 K %

PTM Analysis

Glycosylation: N-linked: PNGFase, which leaves modified N (mass= +1 Da) O-linked: beta-eliminate O-glycosylations with NH4OH, which

leaves modified S and T (mass= -1 Da)

PTM Analysis Flowchart

ptm-report

Mike Coleman

contrast-ptm

Mike Coleman

Time-line

Sear

ch

LC/L

C-M

S/M

S

Dig

estio

n

DTA

Sele

ct

1 w

eek

2 w

eeks

CO

NTR

AST

PTM

s R

esul

ts

3-6

Mon

ths

PTM

s Se

arch

es

1 M

onth

Technology & Methods Seminar “Blast and Other Methods of Probabilistic Sequence Comparison”...

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Transcript of Technology & Methods Seminar “Blast and Other Methods of Probabilistic Sequence Comparison”...