Oct 2010 SDMBT

Lecture 10Interpretation of Mass Spectra

A.Peptide Mass FingerprintingB.MS/MS sequencing

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General workflow for proteomic analysis

SampleSample preparation

Protein mixture Sample separation and visualisation

Comparative analysis

DigestionPeptides

Mass spectrometry

MS dataDatabase search

Protein identification

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Peptides

Protein separated on 2D-gel

Tryptic digest Virtual Tryptic Digest allknown proteins

Trypsin cuts at C-terminal side of lysine and arginine – size ofpeptides unique for each protein

Peptide Mass Fingerprinting (PMF)

ExperimentallyOn MALDI-TOF

match

Oct 2010 SDMBT

King’s College London

(Pierce)

Major peaks at:

646 742 748

780 830 2186

Recall: Tryptic digest of β-casein

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Peptide Mass Fingerprinting (PMF)

Amino acid Sequence fromGenBank

http://www.ncbi.nlm.nih.gov/entrez/

Virtual peptide digest

Peptide Mass Fingerprinting (PMF)

http://bioinformatics.org/sms2/genbank_fasta.html

Convert to FASTA format

Peptide Mass Fingerprinting (PMF)Virtual peptide digest

http://www.expasy.org/

Peptide Mass Fingerprinting (PMF)Virtual peptide digest

Results of virtual tryptic digest

Peptide Mass Fingerprinting (PMF)Virtual peptide digest

Major peaks at:

646 742 748

780 830 2186

Compare with experimental peaks in MALDI-TOF of tryptic digest

Oct 2010 SDMBT

Peptide mass fingerprinting (PMF)Peptide masses are matched against theoretical digests of proteins in databases

Matches are ranked by the number of matching peptides

Confidence in the identity is given by

•a large gap in the number of matching peptides between the 1st and 2nd ranked protein

•good coverage of the 1st ranked protein with the experiment results

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Variables for database searchChoice of database (public or private)

Species of origin

Molecular weight and pI range

Enzyme used for digest

Modifications (reduction, alkylation, phosphorylation)

Tolerance

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Peptide Mass Fingerprinting (PMF)

Oct 2010 SDMBT

PMF using MS-FIT

http://prospector.ucsf.edu/

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PMF using MS-FIT

Choice of database Choice of enzyme12

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PMF using MS-FIT

Tolerance

Peaks entered here

Choice of modifications

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Peptide Mass Fingerprinting (PMF) results for tryptic digest of β-casein

Same protein across 4 similar species

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Does this agree with position in 2D-gel?

Peptide Mass Fingerprinting (PMF) results for tryptic digest of β-casein

Note: do not need match all peaks orwhole protein to identify protein!

Oct 2010 SDMBT

Limitations of PMF

This method assumes that databases are completebut the genomes of only some organisms are completely sequenced, high confidence matches might not be available

But homology between organisms allow for good results

No information about amino acid sequence, only identity of protein. The amino acid sequence in slide 15 is only the ‘predicted sequence’ based on virtual digest.

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Peptide Mass Fingerprinting (PMF)

(Promega)17

Database search is only good as the database and the input data e.g.MALDI spectra often have peaks due to trypsin autolysis and keratindegradation

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Real world MS data

(L&T Inc)18

Peptide Mass Fingerprinting (PMF)If the MS is too noisy…..

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Peptide Mass Fingerprinting (PMF)Exercise: Identify this protein

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Fragmentation of peptides causes cleavages along the peptide backbone

Comparison of MS-MS spectra allows in theory determination of possible amino acid sequences manually (slides 21-33)

Sequences matched to databases to determine identity and sequence of proteins (slides 34 onward)

Adds another layer of certainty in the identification of the peptide and hence to the protein

MS/MS sequencing

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Proteins digested into peptides by trypsin

All tryptic peptides have similar structure – because digested by trypsin

N-terminal of peptide

C-terminal always Arginine (R) or Lysine (K)

When peptides ionised usually– 2+ charge on either end of peptide

MS/MS fragmentation of peptide in 6 ways leads to …..

Trypsin cuts C terminal side of R/K

By convention N-terminal on left

TRYPTIC PEPTIDES IN MS/MS

MS/MS sequencing

IMPORTANT

Although 6 possible ways, generally b and y ions are most common

By convention, ion fragments are called….

It is in general not always to predict what sort of ions will be produced

Explain how does ionisation break up?

Left-hand sideN-terminus

Right-hand sideC-terminus

In theory 8 y-ions and b-ions possible but not all may be observed

Residue mass of amino acid

N-terminalResidue mass+1

C-terminalResidue mass+19

In practice, not all y and b ions observed (cannot be predicted)

Just looking at the y ions

y7

y6

y5

y4y3

y2

246.2 303.3 374.2 477.0 534.3 605.3

7157.3102.870.957.1

Ala (A)Gly (G)Cys (C)

Ala (A)Gly (G)

y-ions contain the C-terminus

therefore … AGCAG….CO2H

Difference betw y ions=Residue mass (see next page)

MS/MS sequencing

Oct 2010 SDMBT

Residue masses of amino acids

letter name mass, Da

G glycine 57.02

A alanine 71.04

S serine 87.03

P proline 97.05

V valine 99.07

T threonine 101.05

C cysteine 103.01

I isoleucine 113.08

L leucine 113.08

N asparagine 114.04

letter name mass, Da

D aspartic acid 115.03

Q glutamine 128.06

K lysine 128.09

E glutamic acid 129.04

M methionine 131.04

H histidine 137.06

F phenylalanine 147.07

R arginine 156.10

Y tyrosine 163.06

W tryptophan 186.08

(N.S. Weld)26

Note: some have very similar molecular weights

Residue mass = Molecular weight of amino acid –18 (2xH + 1xO)

Just looking at the b ions

b7

b6

b5

b4b3

b2

170.9 242.0 299.2 402.0 472.5 530.2

57.5

70.3

102.857.271.1

Cys (C)Ala (A) Gly (G)

b-ions contain the N-terminus

therefore … NH2-…….AGCAGA

b8

600.7

70.5

Ala (A)

Gly (G) Ala (A)

MS/MS sequencing

Combine the results…..

from y-ions… …….AGCAG….CO2H

from b-ions … NH2-…….AGCAGA….

Partial sequence - NH2-….AGCAGA….CO2H

Need to know how to interpret MS – which peaks are y- and b-? Which are y2, y3 etc?Difficult to tell the amino acids at the beginning and the end

MS/MS sequencing

am ions – add all m residue masses - 27xn ions – add all n residue masses + 45

cm ions – add all m residue masses +17zn ions – add all n residue masses + 2

ym ions - add all m residue masses + 19bn ions – add all n residue masses + 1

Useful numbers and Hints for MS-MS spectra

MS/MS sequencing

NH3+

NHNH

OH

O

O

OH

CH3

NH3+Where do these numbers come from?

b-ion (b1)

NH2

CH3

C+

O

NH

CH3

O

OH

H

Definition of residue mass of amino acid =Molecular weight of amino acid –18 (2xH + 1xO)

b ion has 1 extra hydrogen Compared to “residue mass of amino acid”

MS/MS sequencing

NHNH

OH

O

NH2

O

H

NH3+

NHNH

OH

O

O

OH

CH3

NH3+

Where do these numbers come from?

y-ion (y2)

NHOH

O

OH

NH2

NH3+

ResidueMass ofGly

ResidueMass ofLys

Residue mass of Gly+Lys + 2xH + 1xH+1xO = sum of residue masses+19

MS/MS sequencing

NH2

NHOHNH2

NH

O

O

Draw the a,b,c and x,y,z ions from this dipeptideand Calculate the m/z ratios

MS/MS sequencing

NH2NH

NH

O

O

O

OH

CH3CH3

CH3

NH2

Draw the a,b,c and x,y,z ions from this tripeptideAnd calculate the m/z ratio

MS/MS sequencing

Oct 2010 SDMBT 34

Fragment peptides

Peptide after ionisation by MALDI or ESI

Fragmentation Virtual Fragmentation

MS/MS sequencing

experimental match

eg peptide from human catalase LSQEDPDYGIR

Protein Prospector – MS-Producthttp://prospector.ucsf.edu/

Paste amino acid sequence

All predicted a, b, y ions etc.

MS-MS data – amino acid sequence – protein identification

e.g. if MS-MS of aA peptide of mass 1292.61has the following peaks1179.53 1092.50 964.44 835.39 720.37 623.31 508.29 345.22 288.20 175.12

First number - must be mass of peptide+1 i.e. [M+H]+

In ESI-MS tryptic peptide is usually 2+ – it is actually [M+2H]2+

Output – protein identified

MS/MS sequencing

Each of the fragments identified as y or b ions – the user does nothave to assign the peaks or work out residual masses

MS/MS sequencing

More complex example…..

MS-MS of a peptide with mass 1217.58 with peaks at

1088.54 975.46 847.40 746.35 631.32 457.28 358.21243.13 300.16 371.19

Yeast alcohol dehydrogenase – But deliberately missed out one y ion andall except 3 b ions

MS/MS sequencing

All peaks identifiedas y or b ions

Still able to identify the protein.Even though info incomplete