How to identify peptides
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How to identify peptides. Gustavo de Souza IMM, OUS. October 2013. Peptide or Proteins?. Bottom-up Proteomics. 2DE-based approach. Peptide Mass Fingerprinting. MALDI (Matrix Assisted Laser Desorption Ionization). Peptide Mass Fingerprinting. Intensity. m/z. MS/MS. Ion Source. Mass

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How to identify peptides

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How to identify peptides

How to identify peptides

Gustavo de SouzaIMM, OUS

October 2013


How to identify peptides

Peptide or Proteins?


How to identify peptides

Bottom-up Proteomics


How to identify peptides

2DE-based approach


How to identify peptides

Peptide Mass Fingerprinting

MALDI (Matrix Assisted Laser Desorption Ionization)


How to identify peptides

Peptide Mass Fingerprinting

Intensity

m/z


How to identify peptides

MS/MS

Ion

Source

Mass

Analyzer

Mass

Analyzer

Mass

Analyzer

Detector

Collision cell


How to identify peptides

MS/MS

899.013

899.013

899.013


How to identify peptides

Fragmentation

Nomenclature for peptide sequence-ions:

Collision-Induced Dissociation (CID):

MHnn+* + N2 --> b + y

Electron Capture Dissociation(ECD):MHnn++ e- --> MHn(n-1)+· --> c + z·


How to identify peptides

y

y

y

y

y

y

y

7

6

5

4

3

2

1

O

O

O

O

R

R

R

R

2

4

6

8

H

H

H

OH

H

N

N

N

N

2

N

N

N

N

H

H

H

H

R

R

R

R

1

3

5

7

O

O

O

O

b

b

b

b

b

b

b

1

2

3

4

5

6

7

Fragmentation

Roepstorff-Fohlmann-Biemann-Nomenclature


How to identify peptides

Fragmentation

12 aa

b ions

y ions


How to identify peptides

MS/MS of a peptide

y8

P y++13

VPTVDVSVVDLTVK

y10

y6

y9

b5

y12

y11

y4

y5

y7

b6

b3

b10

y3

b4

b8

b7

P y13

b9

y2

b11

b12

b13


How to identify peptides

How to Identify MS/MS

Stenn and Mann, 2004.

Peptide Sequence Tags

Autocorrelation

Probability based match


How to identify peptides

Submitting to Search


How to identify peptides

Protein database (fasta)

x

x

x

How identification happen?

Your data

Step 1: which theoretical peptides has the same mass of the observed ion?

Step 2: From those, whichone have the most similarfragmentationpattern?


How to identify peptides

High mass accuracy – what is it good for?

All theoretical tryptic peptide masses from human IPI database

Example Tryptic HSP-70 peptide: ELEEIVQPIISK, mass 1396.7813 Da

Instrument

LTQ

QSTAR

QSTAR

LTQ-FT

LTQ-FT

LTQ-FT

Mass Accuracy

500

20 ppm

10 ppm

2 ppm

1 ppm

0.5 ppm

Calibration

Ext.

Ext.

Int.

Ext.

Ext-SIM

Int.

# of tryptic peptides for m/z 1396.7813

344

52

33

11

9

3


How to identify peptides

Defining the “Search Space”


How to identify peptides

The “Search Space”

2 mcl

1 mcl

0 mcl

1/2/3

1/2

2/3

2/3/4

4/5

3/4/5

1

3/4

4/5/6

3

5/6

2

4

5

1/2

2/3

1

6

3

2

4/5

4

3/4

5

5/6

6

1

3

2

4

5

6


How to identify peptides

Importance of Search Space Size

Search tool does notidentifya peptide. It only reports the statiscally most suitable theoretical sequence related with the experimental data.

If you increase the size of the database too much, or the size of the search space, false-positive rates also increase.


How to identify peptides

Defining FDRs

Steen and Mann, 2004


How to identify peptides

MOWSE

Chance that two peptides with different sequences but approximate Mr and sharing MS/MS similarities.

More variables inserted during search  Higher chance to get random events  Higher MOWSE score threshold

  • Parameters that can modify the MOWSE calculation:

  • Database size;

  • MMD (measured mass deviation);

  • Number of PTMs choosen;

  • Data quality.


How to identify peptides

Probability Based Mowse Score

Ions score is -10*Log(P), where P is the probability that the observed match is a random event.Individual ions scores > 7 indicate identity or extensive homology (p<0.05).Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.

Example of MMD issue

  • Mycoplasma sp. sample (Munich 2006):

  • Database had ~ 700 entries;

  • Data accuracy had 0.7ppm average;

  • MMD used during search: 3 ppm.


How to identify peptides

Strategies to Visualize FDRs

Peng et al (2003). Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Prot Res 2, 43-50.

Reversed database sequence


False positive identification using reversed database

False positive identification using reversed database


How to identify peptides

Typical Result


How to identify peptides

How to Validate the Data

Are there any Reversed hit protein with 2 peptides above MOWSE score?

-No: All proteins identified with 2 peptides score higher than p<0.05 are good

-Yes: Repeat mascot search with more stringent parameters.

What about 1-hit wonders? (Proteins identified with only 1 peptide)


How to identify peptides

How to Validate the Data

Basically, the idea is to ”play around” with the statistics to make your result more reliable.


How to identify peptides

Take home message

  • Data quality (mass accuracy) and a well-defined search space are key for reliable peptide identification

  • Reliable identification is an interplay between asking enough without asking too much (careful when trying to get “as many IDs as I can”!)


How to identify peptides

PTMs

Gustavo de SouzaIMM, OUS

October 2013


How to identify peptides

PTMs in biology


How to identify peptides

PTMs in biology


How to identify peptides

Complexity of Protein Samples in Eukaryotes

Modifications are specificto a group of amino acids


How to identify peptides

What difference to expect at MS level?

Larsen MR et al, 2006.


How to identify peptides

Defining the “Search Space”


How to identify peptides

PTM abundance in a cell

Total peptides in a sample

Modified peptides

Number of Peptides

Abundance level

Differences from 10e2 to 10e4


How to identify peptides

PTM abundance in a cell


How to identify peptides

Stable vs. Labile PTMs

Larsen MR et al, 2006.


How to identify peptides

Neutral loss

Boersema PJ et al, 2009.


How to identify peptides

Identifying Labile PTMs

Larsen MR et al, 2006.


How to identify peptides

HCD fragmentation

Larsen MR et al, 2006.


How to identify peptides

Status of PTM coverage

Lemeer and Heck, 2009.


How to identify peptides

Status of PTM coverage

Derouiche A et al, 2012.


How to identify peptides

Take home message

  • Depending on PTM, identification can be very easy or very hard

  • Dependent on stability under fragmentation and abundance in the sample

  • ID improvement was mostly defined by instrumentationimprovements (sensitivity etc)


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