Kathleen Sanders BCHM 676 Presentation

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Kathleen Sanders BCHM 676 Presentation

• A Novel Quantitative Proteomics Strategy To Study
• Phosphorylation-Dependent Peptide-Protein
  Interactions
• Fei Zhou, Jacob Galan, Robert L. Geahlen, and W.
  Andy Tao
• Department of Medicinal Chemistry and Molecular
  Pharmacology, and Department of Biochemistry and
• Bindley Bioscience Center, Purdue University,
  West Lafayette, Indiana
• Journal of Proteome Research, 2006

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Overview

• I. Introduction
• Cell Signaling via protein-protein
  interactions
• Syk protein tyrosine kinase
• iTRAQ
• II. Materials and Methods
• Phosphopeptide Affinity Isolations
• iTRAQ Peptide Labeling
• LC Sample Isolation and MS Data Acquisition
• III. Results/Discussion
• Strategy to study multiply phosphorylated
  peptide-protein interaction sites
• Identification of Syk Interacting Proteins
• Findings and Interpretations
• Technique Strengths and Limitations
• V. Conclusion

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IntroductionRegulation of Cell Signaling via
Protein-Protein Interactions

• PTMs
• Syk protein tyrosine kinase
• 72 kDa nonreceptor protein tyrosine kinase
• N-terminal tandem pair of SH2 domains
• C terminal catalytic domain
• Long linker B region
• ? PY342, PY346

? Phosphorylation
?
regulation of proteinprotein interactions
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Schematic structure of Syk/ZAP-70 family protein
tyrosine kinases
The tandem SH2 domains and the kinase domain are
shown as orange and green cylinders respectively.
Purple tubes represent the linker region
connecting the SH2 and kinase domains. In
comparison with the alternatively spliced isoform
Syk-B, Syk contains an insert of 23 amino acids
(dark purple) within the linker region. Tyrosine
residues (Y) in Syk which have been shown to
undergo phosphorylation are indicated these may
be important in regulating enzymatic activity or
recruiting other signaling proteins (Turner et
al., 2000).
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IntroductionSyk Protein Kinase

• Numerous sites of tyrosine P on Syk
• Y342 and Y346 optimal signaling
• SH2 domains that bind to
• one P site/ the other/ or both
• Critical role in immune cells
• Coupling immune recognition receptors
• Multiple signaling pathways

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IntroductionSyk Signaling Pathway
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IntroductioniTRAQ

• iTRAQ isobaric Tagging for Relative and
  Absolute Quantification
• Analyze 4 samples simultaneously
• Examine syk Y342 and Y346 interactions in
  parallel at different P states
• Y342 and Y346 baits in affinity pull down
  experiments

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Materials and MethodsPhosphopeptide Affinity
Isolations

• 4 syk-derived peptides with the two tyrosine
  residues Y342 and Y346 differentially P
• YY DTEVYESPYADPEEIR
• pYY DTEVpYESPYADPEEIR
• YpY DTEVYESPpYADPEEIR
• pYpY DTEVpYESPpYADPEEIR

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Materials and MethodsPhosphopeptide Affinity
Isolations

• Syk peptides (containing C-terminal amines) were
  covalently coupled to a gel through the
  N-terminal amine
• DG75 B cells were cultured and lysed
• Unbound proteins adsorbed onto (20ul of) each
  peptide-residue
• Bound proteins eluted digested w/trypsin
• Resulting peptides labeled w/iTRAQ 114-117
  reagents
• (YY ? 114, pYY ? 115, YpY ? 116, pYpY? 117)

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Materials and MethodsPhosphopeptide Affinity
Isolations iTRAQ Labeling
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IntroductioniTRAQ Review
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Materials and MethodsPhosphopeptide Affinity
Isolations iTRAQ Labeling
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Materials and MethodsLC sample Isolation and MS
data Aquisition

• Peptides separated by microcapillary LC (Agilent)
• MALDI-TOF/TOF mass spec (ABI 4700)
• Alpha-cyano-4-hydroxycinnamic acid matrix
• Syringe pump (Harvard Apparatus) at 800 nl/min
• Mixed postcolumn w/ LC effluent (600 nl/min) via
  Upchurch T connector
• Spotted on 144-spot MALDI target every 20 s
  during 60 min gradient
• SEQUEST used to search MS/MS data
• Mass tolerance 0.1Da
• Fixed modifications Lys and N-termini
• Peptide and probability scores (data filter)
  0.9 and 0.8
• Libra program (quantitative info)

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ResultsSDS-PAGE Analysis of Proteins Isolated
from Peptide Affinity Purification
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(No Transcript)
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Results
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Results
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Results of Data Base Search

• m/z 1189.7 YLYVAAVFR Tubulin B-2 chain
• m/z 1816.8 YSYYDESQGEIYR C2orf4
• Quantitative information was indicated by 4 low
  mass signature ions (m/z 114-117)
• Tubulin B-2 chain 1111 ratio of 114-117
• ? background or nonspecifically adsorbed protein
• C2orf4 binds preferably to P Y346

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• Table 1. A Partial List of Proteins Identified
  in Immobilized Peptide Affinity Pull-Downs
• IPI no. protein
  average ratio
  (114115116117) peptide
  sequence
• IPI00290770 TCP-1-gamma
  10.921.081.07
  TAVETAVLLLR
• IPI00290770 TCP-1-gamma
  
  ISDLAQHYLMR
• IPI00465248 Enolase
  1 10.780.780.96
  YISPDQLADLYK
• IPI00465248 Enolase 1
  
  
  AAVPSGASTGIYEALELR
• IPI00465248 Enolase 1
  
  
  TASAGIQVVGDDLTVTNPKR
• IPI00465248 Enolase 1
  
  IGAEVYHNLK
• IPI00465248 Enolase 1
  
  LAQANGWGVMVSHR
• IPI00465248 Enolase 1
  
  SGKYDLDFKSPDDPSR
• IPI00397984 Splice Isoform of SET protein
  10.851.561.23
  IDFYFDENPYFENK
• IPI00442522 L-lactate dehydrogenase A chain
  10.951.390.94
  GEMMDLQHGSLFLR
• IPI00442522 L-lactate dehydrogenase A chain
  
  FIIPNVVK
• IPI00442522 L-lactate dehydrogenase A chain
  
  LKGEMMDLQHGSLFLR
• IPI00012507 GTP-binding nuclear protein RAN
  10.850.850.96
  YVATLGVEVHPLVFHTNR
• IPI00012507 GTP-binding nuclear protein RAN
  
  KYVATLGVEVHPLVFHTNR
• IPI00383758 Glyceraldehyde-3-phosphate d.
  10.800.820.83
  VVDLMAHMASKE

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• Table 1. A Partial List of Proteins Identified
  in Immobilized Peptide Affinity Pull-Downs
• IPI no. protein
  average ratio (114115116117)
  peptide sequence
• PI00012048 Nucleoside diphosphate kinase A
  11.051.100.98 DRPFFAGLVK
• IPI00012048 Nucleoside diphosphate kinase A
  
  VMLGETNPADSKPGTIR
• IPI00000874 Peroxiredoxin 1
  11.301.891.56
  LVQAFQFTDK
• IPI00000874 Peroxiredoxin 1
  
  ATAVMPDGQFK
• IPI00000874 Peroxiredoxin 1
  
  GLFIIDDKGILR
• IPI00000874 Peroxiredoxin 1
  
  KQGGLGPMNIPLVSDPKR
• IPI00000874 Peroxiredoxin 1
  
  TIAQDYGVLKADEGISFR
• IPI00003362 78 kDa glucose-regulated protein
  10.790.890.85
  AKFEELNMDLFR
• IPI00003362 78 kDa glucose-regulated protein
  
  IEIESFYEGEDFSETLTR
• IPI00003362 78 kDa glucose-regulated protein
  
  IINEPTAAAIAYGLDKR
• IPI00025252 Protein disulfide-isomerase A3
  10.890.920.97
  FLQDYFDGNLKR
• IPI00025252 Protein disulfide-isomerase A3
  
  FVMQEEFSR
• IPI00217223 Multifunctional protein ADE2
  10.791.001.05
  EIVLADVIDNDSWR
• IPI00217223 Multifunctional protein ADE2
  
  IKAEYEGDGIPTVFVAVAGR
• IPI00023860 Nucleosome assembly protein 1
  10.950.950.99
  LDGLVETPTGYIESLPR

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ResultsIdentification of peroxiredoxin 1 binding
to Syk peptide preferably when Y346 is
phosphorylated
The author claims this is a pattern similar to
and confirmatory of that revealed by quantitative
MS.
PD 1 was present in samples purified by both YpY
and pYpY and in greatest abundance in sample YpY
To verify the specificity of this interaction,
the presence of PD 1 among the proteins that
bound to each immobilized peptide was examined by
western blotting
Concerned by the relative intensities of the 114
and 115 peaks
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DiscussionTechnique Strengths and Limitations

• Strengths
• The use of iTRAQ reagents to gain accurate
  quantitative information in the MS/MS spectra has
  an advantage over other stable isotopic labeling
  methods which provide quantitative information in
  the MS spectra
• MS spectra are more complicated and have a much
  higher noise level than MS/MS spectra, making
  accurate measurements of the levels of
  differentially labeled low-abundance peptides
  difficult
• Limitations
• Only a few proteins bound preferentially to P
  Y342
• Not due to an improperly immobilized peptide
  (Western blotting analysis confirmed
  preference of Vavl for binding to pYY peptide)
• The immobilization of peptides via N-termini on
  the solid phase may selectively decrease binding
  to pY342 due to steric hindrance

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Conclusion

• iTRAQ reagents w/4 isobaric tags were employed to
  investigate the role of multiple sites of
  modification in protein-protein interactions
• Tags were used that generate signature ions under
  MS/MS conditions optimal for peptide
  fragmentation
• 76 proteins were identified, 11 dependent on the
  P status of individual tyrosines
• The use of 4 fold multiplexing to enable relative
  protein measurements simultaneously and to
  distinguish interacting proteins dependent on the
  P of specific residues was demonstrated

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References

• Zhou, Galan, Geahlen, Tao, A Novel Quantitative
  Proteomics Strategy To Study Phosphorylation-Depen
  dent Peptide-Protein Interactions. Journal of
  Proteome Research. 2007, 5, 133-140
• Karas, M. Bahr, U., Laser Desorption Ionization
  Mass Spectrometry of Large Biomolecules. Trends
  Anal. Chem. 1990, 9, 321-325.
• Ross, Huang, Marchese, Williamson, Parker,
  Hattan, Khainovski, Pillai, Dey, Daniels,
  Purkayasthan,Juhasz, Martin, Bartlet-Jones, He,
  Jacobson, Pappin, Muttiplexed Protein
  Quantitation in Saccharomyces cerevisiae Using
  Amine-reactive Isobaric Tagging Reagents.
  Molecular Cellular Proteomics. 2004,
  31154-1169
• www2.hawaii.edu/_johnnb/micro/micro161/T_andB
• Alberts, B., The Molecular Biology of The Cell
  (fourth edition), 2002, Garland Science.