Download Protein Assignment

Introduction to Mass Spectrometry-based
Proteomics
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Central dogma of molecular biology
DNA
mRNA
transcription
Protein
translation
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Alternative Splicing
exon3
exon2
exon1
exon4
IAMTHEQSRTV FIRST QPBASRTVSECONDQABDECTV ALTERNATIVE
intron1
intron2
intron3
IAMTHEFIRSTALTERNATIVE
I AM THE FIRST ALTERNATIVE
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Alternative Splicing
exon3
exon2
exon1
exon4
IAMTHEQSRTV FIRST QPBASRTVSECONDQABDECTV ALTERNATIVE
intron1
intron2
intron3
IAMTHESECONDALTERNATIVE
I AM THE SECOND ALTERNATIVE
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Proteomics
Post-translational
Modification (PTM)
conformational
change
change in
cellular localization
protein-protein
interaction
Various
Cellular Functions
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Genomics vs. Proteomics
Blueprint vs. Machine
DNA
Proteins
Easily amplified by PCR
No amplification technique
Can be “unfolded” and
“refolded” reversibly
Folded structure very
sensitive to conditions
Chemically simple –
negatively charged
backbone, 4 nucleotides
Chemically complex –
20 amino acids of non-polar,
polar, charge, or
hydrophobic character
The amount of DNA is
“stable” within a cell
Protein concentration
within a cell are highly
variable
DNA molecules tend not to
associate
Proteins often form
complexes
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Proteomics: study of protein
Protein
expression
Proteinprotein
interaction
Protein
structure
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Proteomics
http://www.childrenshospital.org/research/_proteomics
/index.html
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Sample - Protein Digestion
Protein
 An amino acid sequence
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRKL
HQEDNDYINASLIKMEEAQRSYILTQ….
Peptide
 Substring of the protein.
LPKNKNRNRYRDVSPFDHSR
Trypsin
 Aggressive and stable protease
 It cleaves proteins very specifically on the carboxy-terminal side
(right side) of arginine(R) and lysine(K) residues.
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Sample - Protein Digestion
Miscleavage(Faulty cleavage)
Semitryptic
Nontryptic
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Proteomics
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Separation - GEL
Separation by 2DE gels
Basic scheme
1.
separation by pI and mw
2.
stain gel (coomassie, silver)
3.
semi-quantitation based on spot
size/intensity
Advantages
1.
great for visualizing whole-cell proteome
2.
useful for comparative proteomics
Disadvantages
1.
low expression protein often not visible
2.
cannot be automated
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Separation – LC
Liquid chromatography is a separation technique that
involves:
 injection of a small volume of liquid sample
 into a tube packed with porous particles (stationary phase)
 where individual components of the same sample are transported
along the packed column by a liquid moved by gravity.
The components of the sample are separated from one
another by the column packing that involves various
chemical and/or physical interactions between the
molecules and the packing particles.
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Separation - HPLC
High-Performance LC is a separation technique that
involves:
 injection of a small volume of liquid sample
 into a tube packed with tiny particles (3-5 micron in diameter called
stationary phase)
 where individual components of the same sample are moved down
the packed column with a liquid (mobile phase) forced through the
column by high pressure delivered by pump.
The components of the sample are separated from one
another by the column packing that involves various
chemical and/or physical interactions between the
molecules and the packing particles.
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Proteomics
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Mass spectrometry
Hanyang Univ. 16
Mass spectrometry
2002
Nobel Prize
Soft ionization
MALDI
matrix-assisted laser desorption
Koichi Danaka
ESI
electrospray ionization
John Fenn
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Mass Spectrometry
Ion selection
Quadrupole
https://www.youtube.com/watch?v=qxPb9vFWdqo
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Mass Spectrometry
TOF Mass analyzer
ion selection
•
Ion trap
•
Quadrupole
ion detection
•
Time-of-flight (TOF)
•
Fourier-Transform Ion Cyclotron
Resonance MS
(FT-ICR MS)
Common:
masses separated by m/z
a.
b.
c.
d.
ion source
reflectron
ion path
detector
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Mass Spectrometry
TOF Mass analyzer
m/z can be inferred from time spent in flight.
The same force will accelerate each ion differently based on its
mass (m) and charge (z).
++
+
Detector
+
+
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Mass Spectrometry
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Single stage MS
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Tandem MS
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MS vs. MS/MS
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Mass vs. Intensity vs. Time
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The mass-spectrometry/proteomic experiment
Peptide mixture
HHHH
Protein mixture
EEEE
FFFF
KKKK
QQQQ
HHHH
SSSS HHHH
AAAA
CCCC RRRR TTTT
SSSS
AAAA LLLL AAAA
KKKK
CCCC
QQQQ EEEE
PPPP PPPP
HHHH DDDD
IIII
FFFF GGGG
IIII FFFF
TTTT MMMM
HHHH
digestion
IIIIPPPPMMMMGGGGHHHHIIII
MMMM
mass-spectrometry
m/z
m/z
m/z
intensity
intensity
intensity
m/z
KKKK
LLLL
SSSS
intensity
m/z
MMMM
PPPP
QQQQ
intensity
intensity
intensity
AAAA
CCCC
DDDD
intensity
intensity
Mass spectra
m/z
m/z
m/z
m/z
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The mass-spectrometry/proteomic experiment
Protein assignment
Peptide assignment
HHHH
KKKK
EEEE
Peptide assembly
IIIIPPPPMMMMGGGGHHHHIIII
HHHH
FFFF
QQQQ
HHHH
MMMM
SSSS HHHH
RRRR
AAAA
CCCC
TTTT
SSSS
AAAA LLLL AAAA
KKKK
CCCC
QQQQ EEEE
PPPP PPPP
HHHH DDDD
IIII
FFFF GGGG
IIII FFFF
TTTT MMMM
Peptide validation
Experimental mass spectra
Peptide-spectrum match
KKKK
intensity
intensity
m/z
MMMM
intensity
intensity
AAAA
intensity
intensity
Database search
De novo sequencing
m/z
m/z
m/z
m/z
m/z
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Pipeline : integrated tools for MS/MS proteomics
Input
Spectrum data
(Protein database)
Peptide assignment
SEQUEST
PEAKS
MODi
Quantitation
ASAPRatio
MaxQuant
Peptide validation
manual validation
PeptideProphet
Target/Decoy
Protein assignment
& validation
ProteinProphet
IDPicker
Output
Interpretation
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intensity
Peptide assignment
m/z
>Protein A
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFD
HSRKLHQEDNDYINASLIKMEEAQRSYILTQQIDKSGSWAAIYQDIRHEASDF
HEASDFPCRVAKLPKNKNRNRYMEKEFEQIDKSGSWAAIYQDIRHEMEKEFEQ
IDKSGSWAAIYQDIRHE
>Protein B
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQ
QQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPE
VMGVSKVKEAMAPKHKEMPFPKYPVEPF
>Protein C
...
VAKLPKNKNR
...
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Peptide validation
Peptide-spectrum match
CCCC
GGGG
intensity
intensity
intensity
AAAA
m/z
m/z
m/z
LLLL
TTTT
intensity
intensity
intensity
KKKK
m/z
m/z
m/z
intensity
IIII
intensity
QQQQ
intensity
LLLL
m/z
m/z
m/z
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Protein Assignment
Peptides
Proteins
HHHH
EEEE
KKKK
FFFF
QQQQ
HHHH
SSSS HHHH
AAAA
CCCC RRRR TTTT
SSSS
AAAA LLLL AAAA
KKKK
CCCC
QQQQ EEEE
PPPP PPPP
HHHH DDDD
IIII
FFFF GGGG
IIII FFFF
TTTT MMMM
HHHH
IIIIPPPPMMMMGGGGHHHHIIII
MMMM
Missing values
Mass spectra
m/z
m/z
MMMM
m/z
intensity
intensity
PPPP
intensity
IIII
intensity
QQQQ
intensity
LLLL
m/z
m/z
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Protein Assignment
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