the effect of ms/ms fragment ion tolerance on peptide ... · page 10 february 28, 2008 spectrum...

Group/Presentation TitleAgilent Restricted

Month ##, 200X

The Effect of MS/MS Fragment Ion Tolerance

on Peptide Identification in

Shotgun Proteomics

Christine MillerSenior Application Scientist

February 28, 2008

Accurate Mass MS/MSAgilent eseminar

February 28, 2008

Overview

• Agilent 6510 Q-TOF overview

• HeLa cell 2D OFFGEL electrophoresis/HPLC-Chip experimental setup

• Comparison of Ion Trap and QTOF Spectrum Mill search results

• Reverse database searches using different precursor and product ion mass tolerances

• Effect of post-translational modifications and database size on the confidence in peptide identification using different precursor and product ion mass tolerances


February 28, 2008

Experimental Setup

• HeLa sample– 300 µg of trypsinized proteins from a HeLa cell extract was separated

into 23 fractions on an Agilent 3100 OFFGEL Fractionator (pH 3-10)– Approximately 5% of each fraction (5 µL) was analyzed by HPLC-Chip

Q-TOF and the Agilent Ion Trap

• Spectrum Mill– All data analyzed using Spectrum Mill– IPI Human database, trypsin specificity, 2 missed cleavages– Manual validation using Protein Summary modes– Results exported into Microsoft Excel and score distributions plotted as

histograms


February 28, 2008

Agilent 6510 Q-TOF LC/MS

Quickly captures the most information from complex samples

• 10X sensitivity advantage (attomole MS and MS/MS sensitivity)

• Wide in-scan dynamic range covers 3.5 decades

• Fast scan rate (20/sec) at up to 15,000 Resolving Power

Easy and confident identification of unknowns

• 2X typical mass accuracy (1 to 3 ppm MS and 5 ppm MS/MS)

• Automated reference mass introduction and calibration

• Autotune saves time and enhances performance

Ion-trap sensitivity, speed, and accurate mass that redefines the QTOF category


February 28, 2008

3100 OFFGEL FractionatorNew Depth and Speed

Fractionates proteins and peptides by isoelectric point (pI)

pH gradient: immobilized (IPG gels)

Number of samples: 2 trays, 8 strips/tray

Number of fractions: 12 or 24

Fraction volume: 150 µl

Resolution: 0.1/0.5 pH


February 28, 2008

Elements Of The HPLC-Chip

Mass spec spraying needle, needle assembly & fittings

Nano LC Column

Enrichment column, capillaries, fittings, frits

Encapsulated HPLC-Chip for use with Chip cube

HV ESI contact

RF tag


February 28, 2008

TIC of OFFGEL Fraction 4 of Trypsin-Digested HeLa Cell Extract

6x10

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

5

5.25

+ TIC MS(all) HeLa-oge-fr-4.d

1 1

Counts vs. Acquisition Time (min)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88


February 28, 2008

Note: Not all countedion types shown in table

12.004 x 0.25 = 1.000 x 0.25 = 0.000 x 0.25 = 0.001 x .50 = 0.507 x 1.50 = 10.501.50Y++ , y+++

Total positive0.25b-H2 O, y-H2O0.25b-NH3, y- NH3

0.25a ions0.50b ions

12.00 - .028 = 11.97

Score

Points subtracted for unidentified

peak

2.8% y++12

Spectrum Mill Scoring Overview

, y


February 28, 2008

Reverse Database Score

To provide the best score for a wrong answer all database sequence candidates passing parent mass filter are additionally subject to “inner sequence reversal” and interpreted against the MS/MS spectrum.

i.e.SAMPLERBecomes

SELPMAR


February 28, 2008

Spectrum Mill Search Parameters and Results for 2D OFFGEL HPLC-Chip Data

Ion Trap Q-TOF

Total # spectra acquired 355,136 363,692

MS/MS acquired 271,507 186,049

# of extracted files (.pkl) 92,081 129,752

# of search result files (.spo) 72,028 20,871

# of validated spectra 10,618 16,072

# of validated unique peptides 4151 6253

# of protein ID’s 873 994

# of unvalidated spectra 61,410 4799

# of peptide ID’s w/ no rank 2 hit 1454 10453

# of peptides w/ no reverse DB hit 2110 10249


February 28, 2008

Distribution of Mass Errors for Agilent Q-TOF

-20-16 -12 -8 -4 0 4 8 12 16 20ppm error

Precursor

-50-40 -30-20 -10 0 10 20 30 40 50ppm error

Fragment


February 28, 2008

Not validValidQTOF

Ion trap

# of

mat

ches

Spectrum Mill Score

Valid vs. “Not valid” Hits For Q-TOF and Ion Trap

0100200300400500600700800

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

0

1000

2000

3000

4000

5000

6000


February 28, 2008

False Positive Rates Using Reverse Databases

• Comparison of forward and reverse database searches can be used to determine whether or not a match is likely a false positive

– Spectrum Mill delta Forward-Reverse score.– “Target-decoy” approach, Gygi et al

• We employed a modified form of this approach to estimate the false positive rate at different mass tolerances

– Assume forward results shown previously are nominally constant with varied mass tolerance

– Perform reverse database searches against full SwissProt database using a series of different precursor and product ion mass tolerances, and compare these reverse search results with the previous forward search results


February 28, 2008

False Positive Rate Decreases With Improving Precursor Mass Accuracy

Fragment mass tolerance = 40 ppm

0

200

400

600

800

1000

1200

0 2 4 6 8 10 12 14

500 ppm 200 ppm

100 ppm50 ppm

1000 ppm

20 ppm10 ppm

5 ppm

Spectrum Mill Reverse Database Search Score

# of

fals

e po

sitiv

e m

atch

es


February 28, 2008

False Positive Rate Decreases With Improving Precursor Mass Accuracy

Fragment mass tolerance = 40 ppm

0

200

400

600

800

1000

1200

4 4.5 5 5.5 6 6.5

5 ppm10 ppm20 ppm50 ppm100 ppm200 ppm500 ppm1000 ppm


# of

fals

e po

sitiv

e m

atch

es

7


February 28, 2008

False Positive Rate Dramatically Decreases With Improving Fragment Mass Accuracy

0

500

1000

1500

2000

2500

3000

3500

4000

0 2 4 6 8 10 12 14

1000 ppm

500 ppm

200 ppm

100 ppm50 ppm

20 ppm10 ppm

5 ppm

# of

fals

e po

sitiv

e m

atch

es


Precursor mass tolerance = 10 ppm


February 28, 2008

False Positive Rate Dramatically Decreases With Improving Fragment Mass Accuracy

# of

fals

e po

sitiv

e m

atch

es


Precursor mass tolerance = 10 ppm

0

500

1000

1500

2000

2500

3000

3500

40005 ppm10 ppm20 ppm50 ppm100 ppm200 ppm500 ppm1000 ppm

4 4.5 5 5.5 6 6.5 7

False positive rate dramatically increases with increased fragment mass tolerance


February 28, 2008

Reverse Database Searches For Three Precursor and Product Ion Mass Tolerances

Spectrum Mill Reverse Database Score

1000 ppm precursor, 1000 ppm fragment

0

1000

2000

3000

4000

5000

6000

7000

8000

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

# of

fals

e po

sitiv

e m

atch

es


February 28, 2008


5 ppm precursor, 1000 ppm fragment1000 ppm precursor, 1000 ppm fragment

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

1000

2000

3000

4000

5000

6000

7000

8000

# of

fals

e po

sitiv

e m

atch

es



February 28, 2008


# of

fals

e po

sitiv

e m

atch

es

Agilent Q-TOF (10 ppm precursor, 40 ppm fragment)5 ppm precursor, 1000 ppm fragment1000 ppm precursor, 1000 ppm fragment

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

1000

2000

3000

4000

5000

6000

7000

8000



February 28, 2008

Confident Peptide Matches at 1000 ppm Precursor and 1000 ppm Fragment

Spectrum Mill Score

Mass Tolerance (ppm) # of valid matches with displayed confidence

Precursor Fragment >95% >75% >50%1000 1000 954 2220 3361

ReverseForward

5%25%50%

010002000300040005000600070008000

0 2 4 6 8 10 12 14 16 18 20 22 24


February 28, 2008

Confident Peptide Matches at 5 ppm Precursor, 1000 ppm Fragment


Precursor Fragment >95% >75% >50%1000 1000 954 2220 33615 1000 4181 6590 7645

5%25%50%

0

500

1000

1500

2000

2500

3000

3500

Spectrum Mill Score0 2 4 6 8 10 12 14 16 18 20 22 24

ReverseForward


February 28, 2008

Confident Peptide Matches at 10 ppm Precursor, 40 ppm Fragment

5%25%

50%


Precursor Fragment >95% >75% >50%1000 1000 954 2220 33615 1000 4181 6590 764510 40 6590 11552 15509

0100200300400

500600700800

Spectrum Mill Score0 2 4 6 8 10 12 14 16 18 20 22 24


February 28, 2008

Typical Peptide Match at 95% Confidence for 1000 ppm, 1000 ppm


February 28, 2008



February 28, 2008

Number of Protein IDs with a Minimum of 2 Peptides with a Threshold Peptide Confidence

ppm Tolerance # of Valid Protein Matches with Peptide % Confidence (minimum of 2 or more unique

peptides/protein)

Precursor Fragment >95% >75% >50%

1000 1000 102 213 282

5 1000 338 478 524

10 40 478 728 829

Accurate MS/MS data produces a substantial increase in the number of protein IDs with a given confidence


February 28, 2008

Effect of Fragment Mass Tolerance Using More Complex Searches• Determine the effect of precursor and fragment tolerance relative to database size

• Use 3 of the OFFGEL electrophoresis fractions (2, 4, and 6)

• Search 3 different reversed databases:– IPI Human – 68000 entries– IPI Human with variable phosphorylation (up to 2 phosphorylation sites)– NCBInr (mammalian subset) – 470000 entries

• Compare reverse database search results to forward search results and calculate # of ID’s for given false positive rates


February 28, 2008



1000 1000 338 534 771

5 1000 771 1201 1354

10 40 1201 2282 2749



1000 1000 183 338 429

5 1000 534 905 1033

10 40 1201 2040 2676

IPI Human only:

IPI Human + variable phosphorylation:

Effect of Fragment Mass Tolerance for Searches With Post-Translational Modifications


February 28, 2008

Effect of Fragment Mass Tolerance for Larger Databases



1000 1000 183 429 534

5 1000 641 905 1033

10 40 771 1776 2676

NCBInr Mammals:



1000 1000 338 534 771

5 1000 771 1201 1354

10 40 1201 2282 2749

IPI Human only:


February 28, 2008

Why Is Fragment Mass Tolerance Important?

• Many more fragment mass measurements than precursor measurements

• Large fragment masses close in mass to the precursor mass and the effect of narrowing the product ion tolerance will have a similar effect to narrowing the precursor ion tolerance

• Background masses in MS/MS data will likely contribute to tandem MS search scores with decreasing fragment ion mass tolerance


February 28, 2008

Conclusions and Future Directions

Fragment mass tolerance has significant impact on false positive rates in proteomics

• High fragment mass accuracy is important when searching large databases and/or searching for post-translational modifications

The new Agilent Q-TOF gives high mass accuracy for both the precursor and product ions with a throughput appropriate for shotgun proteomics

Mascot results do improve with better mass accuracy

Future directionsExplore abundance-based and/or m/z-based dynamic mass tolerance

Further investigate the effect of fragment mass accuracy and incorporate results into Spectrum Mill and other Agilent products.


February 28, 2008

Acknowledgements

Dave Horn

Bryan Miller


February 28, 2008

Wrap-up E-Seminar Questions

Thank you for attending Agilent e-Seminars. Our e-Seminar schedule is expanding every

week. Please check our website frequently at:

www.agilent.com/chem/education

Or register for

to receive regular updates

Stay currentwith e-notes


February 28, 2008BioColumn Separations

January 30, 2008

March 13, 2008, 10:00 a.m. EDTAnalysis of Intact Proteins in Drug DiscoveryDr. Jonathan Williams, GlaxoSmithKline, Research Triangle Park

April 24, 2008, 11:00 a.m. EDTBiomarker Discovery by Targeted and Profiling ProteomicsProfessor Rainer Bischoff, Analytical Biochemistry, University of Groningen

May 8, 2008, 11:00 a.m. EDTProtein Analysis Using CAD/ETD Iontrap Tandem Mass SpectrometryProfessor Ole Norregard Jensen, Protein Research Group at University of Southern Denmark

June 26, 2008, 11:00 a.m. EDTPeptide Quantitation With An Agilent 6410 QQQ SystemNing Tang, Application Scientist, Agilent Technologies, Inc.

Upcoming Proteomics eSeminars

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