oct 2010 sdmbt lecture 10 interpretation of mass spectra a.peptide mass fingerprinting b.ms/ms...
TRANSCRIPT
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Oct 2010 SDMBT
Lecture 10Interpretation of Mass Spectra
A.Peptide Mass FingerprintingB.MS/MS sequencing
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Oct 2010 SDMBT
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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Oct 2010 SDMBT 3
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
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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)
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Amino acid Sequence fromGenBank
http://www.ncbi.nlm.nih.gov/entrez/
Virtual peptide digest
Peptide Mass Fingerprinting (PMF)
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http://bioinformatics.org/sms2/genbank_fasta.html
Convert to FASTA format
Peptide Mass Fingerprinting (PMF)Virtual peptide digest
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http://www.expasy.org/
Peptide Mass Fingerprinting (PMF)Virtual peptide digest
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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
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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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Oct 2010 SDMBT
Variables for database search
Choice 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)
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Oct 2010 SDMBT
PMF using MS-FIT
http://prospector.ucsf.edu/
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Oct 2010 SDMBT
PMF using MS-FIT
Choice of database Choice of enzyme12
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Oct 2010 SDMBT
PMF using MS-FIT
Tolerance
Peaks entered here
Choice of modifications
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Oct 2010 SDMBT
Peptide Mass Fingerprinting (PMF) results for tryptic digest of β-casein
Same protein across 4 similar species
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Oct 2010 SDMBT 15
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!
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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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Oct 2010 SDMBT
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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Oct 2010 SDMBT
Real world MS data
(L&T Inc)18
Peptide Mass Fingerprinting (PMF)If the MS is too noisy…..
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Oct 2010 SDMBT 19
Peptide Mass Fingerprinting (PMF)
Exercise: Identify this protein
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Oct 2010 SDMBT
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
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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
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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
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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)
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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Oct 2010 SDMBT 34
Fragment peptides
Peptide after ionisation by MALDI or ESI
Fragmentation Virtual Fragmentation
MS/MS sequencing
experimental match
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eg peptide from human catalase LSQEDPDYGIR
Protein Prospector – MS-Producthttp://prospector.ucsf.edu/
Paste amino acid sequence
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All predicted a, b, y ions etc.
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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
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First number - must be mass of peptide+1 i.e. [M+H]+
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In ESI-MS tryptic peptide is usually 2+ – it is actually [M+2H]2+
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Output – protein identified
MS/MS sequencing
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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
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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
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All peaks identifiedas y or b ions
Still able to identify the protein.Even though info incomplete