Nevada Proteomics Center

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Nevada Proteomics Center David R. Quilici
Ph.D. University of Nevada, Reno Department of
Biochemistry
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Facilities

• Genomic Solutions Proprep - performs enzymatic
  digests, peptide extraction and cleanup as well
  as MALDI target spotting.
• ABI 4700 MALDI TOF/TOF- capable of high
  throughput protein identification and sequence
  information.
• Thermo Finnigan LCQ Deca XP plus - LC/MS, Protein
  identification, sequence information and Post
  translational identification.
• Michrom Paradigm - automated 2D LC at nanoliter
  flow rates.
• Dionex Probot Spotting robot for LC/MALDI
  applications.
• Thermo Finnigan Polaris Q volatile and small
  (lt650 amu) nonpolar metabolites analysis.

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Genomic Solutions Investigator Proprep
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Robotic Digestions

• Rate 100 (1 to 93 samples using trypsin)
• Sample State
• In-Gel up to three 1mm round pieces
• In-Solution lyophilized or in appropriate
  buffer

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4700 Proteomics Analyzer with TOF/TOF? Optics
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4700 MALDI TOF/TOF

• Resolution up to 20k in reflector mode
• Mass Accuracy
• Internal Calibration gt5 ppm ie - 0.002 Da _at_
  1000 m/z
• External Calibration gt100 ppm ie - 0.1 Da _at_
  1000 m/z
• Sensitivity 1 fmol on target _at_ 1000 m/z

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MALDI TOF/TOF

• Rate 8.00 per spot
• Sample State
• In-gel
• In Solution
• Intact Proteins
• Amount Needed Visible by CBB staining (gt100
  fmol)
• Capabilities/limitations of Instrument
• High throughput protein identification
• Sequence Information

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Thermo Finnigan LCQ Deca XP plus
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LCQ ESI-Ion trap

• Mass Resolution 4000 ppm
• Mass Accuracy 300ppm
• Sensitivity 200 fmol on column

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LC ESI

• Rate 65
• Sample State Aqueous solution with
• lt5 ACN
• lt 0.01 (w/v) detergent concentration
• lt 1 mM salt
• Amount Required gt 200 fmol
• Capabilities/Limitations of Instrument
• Protein Identification
• Sequence Information
• PTM studies

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Michrom Paradigm
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Dionex Probot
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LC MALDI

• Rate 465
• Sample State Aqueous solution with
• lt5 ACN
• lt 0.01 (w/v) detergent concentration
• lt 10 mM salt
• Amount Required gt 100 fmol
• Capabilities/Limitations of Instrument
• Protein Identification
• Sequence Information

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Thermo Finnigan Polaris Q
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Metabolomics

• Rate 18 (per run)
• Sample State Nonpolar solvent
• Amount Required gt10 ng
• Capabilities/Limitations of Instrument
• Small nonpolar metabolites
• Volatiles
• Multiple Columns

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Services

• Robotic In-Gel or In-Solution Digestion
• Protein Identification
• Protein Sequence Information
• Post Translational Modification Identification
• Metabolomic Identification

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PROTEINIDENTIFICATION

• Gel-Based MALDI
• ABI 4700 MALDI TOF/TOF
• LC ESI
• Michrom Paradigm nano-LC
• Finnigan DECA XP ion trap
• LC MALDI
• Michrom Paradigm nano-LC
• Dionex Probot

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Database Comparison

• NCBInr protein database
• vs
• In house EST database

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Protein ID Comparison
SSP Protein NCBI Mowse score EST
Mowse score
3803 alpha-tubulin 220 164
3408 Oxygen Evolving protein of PS II 239 460
3308 Ascorbate peroxidase 84 262
8107 Pathogenesis - related protein 10 300 235
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NCBI Database search Mowse score 220 Protein
ID alpha-tubulin Betula pendula
UNR EST Database search Mowse score
164 Protein ID tubulin alpha chain Prunus
amygdalus
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NCBI Database search Mowse score 239 Protein
ID putative 33kDa oxygen evolving protein
of photosystem II Oryza sativa
UNR EST Database search Mowse score
460 Protein ID similar to oxygen evolving
enhancer protein 1 precursor Bruguiera
gymnorrhiza
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NCBI Database search Mowse score 84 Protein
ID ascorbate peroxidase Arabidopsis thaliana
UNR EST Database search Mowse score
262 Protein ID similar to L-ascorbate
peroxidase cytosolic isozyme maize
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NCBI Database search Mowse score 300 Protein
ID pathogenesis-related protein 10 Vitis
vinifera
UNR EST Database search Mowse score
235 Protein ID pathogenesis-related protein
10 Vitis vinifera
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Data Base Comparison Summary

• The NCBI Database is a useful tool in the
  Identification of Proteins even though the exact
  Protein is not in the Database
• ESTs become extremely useful in Protein
  Identification of species which the genome has
  not been completed

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Protein Sequencing

• Double Enzymatic Digest
• Trypsin and Chymotrypsin
• De novo software tools

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Fragment 1393 MS/MS
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Peptide Fragmentation

• Series of possible fragment ions from sequence -
• three possible sites for backbone cleavage
• ion can be either of the two fragments formed by
  the cleavage

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A1 fragment 1393
y11
y10
y8
y7
y6
y5
y4
y3
G Y D P A L P I I G H L Q
b12
b11
b10
b9
b8
b7
b6
b5
b4
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Protein Sequence Information Summary

• Multiple enzymatic digests are useful in
  producing smaller more manageable peptides
• MS/MS from the 4700 is very useful in De novo
  interpretation

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Phosphorylation Site Mapping
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Software

• Data Base Search Engines
• MASCOT MALDI
• SEQUEST LC-MS
• De novo Tools
• PEAKS
• De novo Explorer
• De novo X

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Data Analysis

• Rate 40 (per hour 1 hour min.)
• Reports
• GPS Explorer
• IdQuest
• Interpreting Reports

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Contact Us

• How to submit samples
• Kathy Schegg
• FA 118
• 784-6337
• schegg_at_unr.edu
• David Quilici
• FA 122
• 784-1590
• quilici_at_unr.edu
• Rebekah Woolsey
• FA 122
• 784-4248
• rebekahw_at_unr.edu
• More information
• http//www.unr.edu/inbre/cores/proteomics/defa
  ult.asp

Nevada INBRE is supported by NIH Grant Number
P20 RR-016464 from the INBRE Program of the
National Center for Research Resources.