High Throughput Protein Domain Elucidation by Limited Proteolysis-Mass Spectrometry
Download
1 / 17

High Throughput Protein Domain Elucidation by Limited Proteolysis-Mass Spectrometry Jeff Bonanno and Xia Gao Structura - PowerPoint PPT Presentation


  • 196 Views
  • Uploaded on

High Throughput Protein Domain Elucidation by Limited Proteolysis-Mass Spectrometry Jeff Bonanno and Xia Gao Structural GenomiX, Inc . Outline. Overview of SGX technology platform Overview of NY and SGX research consortium (NYSGXRC) Mass Spectrometry applications

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'High Throughput Protein Domain Elucidation by Limited Proteolysis-Mass Spectrometry Jeff Bonanno and Xia Gao Structura' - dieter


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Slide1 l.jpg

High Throughput Protein Domain Elucidation by Limited Proteolysis-Mass SpectrometryJeff Bonanno and Xia Gao Structural GenomiX, Inc


Outline l.jpg
Outline Proteolysis-Mass Spectrometry

  • Overview of SGX technology platform

  • Overview of NY and SGX research consortium (NYSGXRC)

  • Mass Spectrometry applications

    • Integration into SGX technology platform

  • High Throughput Limited Proteolysis Mass Spectrometry


Sgx technology platform l.jpg
SGX Technology Platform Proteolysis-Mass Spectrometry


Overview of ny and sgx research consortium nysgxrc l.jpg
Overview of NY and SGX Research Consortium (NYSGXRC) Proteolysis-Mass Spectrometry

  • Vision

    • The NYSGXRC will establish a cost-effective, high-throughput X-ray crystallography platform that serves as a model for the structural biology laboratory of the future.

  • Mission

    • To develop and use the technology for high-throughput structural and functional studies of proteins

  • www.nysgxrc.org


Nysgxrc members l.jpg
NYSGXRC Members Proteolysis-Mass Spectrometry

  • Albert Einstein College of Medicine (AECOM)

  • Brookhaven National Laboratory (BNL)

  • Columbia University (CU)

  • Structural GenomiX, Inc. (SGX)

  • Sloan Kettering Institute (SKI)

  • University of California at San Diego (UCSD)

  • University of California at San Francisco (UCSF)


Ms analysis from gene expression to protein purification l.jpg
MS Analysis from Gene Expression to Protein Purification Proteolysis-Mass Spectrometry

  • Molecular biology

    • Verify expression and protein integrity

    • Provide domain boundary information

  • Fermentation

    • Determine heavy atom incorporation

    • Monitor progression

  • Purification

    • QA/QC on final pools

    • Characterize post-translational modifications

    • Fraction analysis to provide guidance


Methods for domain elucidation l.jpg
Methods for Domain Elucidation Proteolysis-Mass Spectrometry

  • Bioinformatic approach

    • Sequence alignment, e.g. BLAST, Pfam

    • Secondary structure prediction

    • Homology modeling

  • Limited Proteolysis MS

    • Probing protein structure in solution

    • Provide termini information of protein functional domain(s)

    • Provide information on solvent accessible or disordered loop regions (Cohen et al., 1995, Cohen and Chait, 1996, Marcotrigiano et al., 1997, Lee et al., 1996, Xie et al., 1996, Cabral, et al., 1998, Zhang et al., 1997)

  • Hydrogen/Deuterium Exchange MS

    (Pantazatos et al., 2004)



A successful example l.jpg
A Successful Example Proteolysis-Mass Spectrometry

Domain 1

Domain 2

Domain 3

Domain 4

Cterm

1

Full length protein:

Low yield, 2mg/L

High tendency to aggregate

Cannot be concentrated above 1mg/ml

Domain 1 ?

Domain 2

Domain 3

Domain4

4

Cterm - 76

LP construct:

High yield, 30mg/L

Stable overtime

Concentration of 5mg/ml

Protein crystallized


Proteolysis experiment conditions l.jpg
Proteolysis Experiment Conditions Proteolysis-Mass Spectrometry

  • Proteases

    • Trypsin, Lys-C, Chymotrypsin and GluC

  • Buffer condition

    • PH~8, salt and detergent if necessary--Protein native condition

  • Protein concentration

    • ~2mg/ml

  • Time points

    • 5min, 10min, 30min, 1h, 2hrs and 4hrs.

  • Capacity

    • Eight proteins per experiment


Sample preparation for automated maldi ms analysis l.jpg
Sample Preparation for Automated MALDI-MS Analysis Proteolysis-Mass Spectrometry

  • “Thin-Layer” Sample Preparation

    • (Cadene and Chait, 2000)

    • High homogeneity offers high success rate for automated data acquisition. Better than 95% attempts result in satisfactory MS spectrum.

    • Thin-Layer method affords high detection sensitivity, < 10 fmoles.

  • Automated Data Acquisition by Sequence Control from ABI


Data interpretation with paws l.jpg
Data Interpretation with PAWS Proteolysis-Mass Spectrometry


Data assembly by digests reader in batch mode l.jpg
Data Assembly by Digests Reader in Batch Mode Proteolysis-Mass Spectrometry


Throughput l.jpg
Throughput Proteolysis-Mass Spectrometry

  • Total proteolysis experiments: 270

  • Total number of data sets acquired: 250

  • Total number of data sets analyzed: 210

  • Duration of this endeavor: six months

  • Total FTEs: ~1.0 on average


Representative results l.jpg
Representative Results Proteolysis-Mass Spectrometry

  • Summary of MS analysis of crystallized proteins which diffract poorly—Three examples

    • Proteins showed stability toward proteolysis

    • Removal of His6-tag is recommended

    • Removal of structural micro-heterogeneity necessary

  • Large scale cloning, expression/solubility testing and resubmission to crystallization underway


Plans forward l.jpg
Plans Forward Proteolysis-Mass Spectrometry

Automated

Proteolysis

Experiment

Automated

Data

Acquisition

Automated

Data

Assembly

Automated

Molecular

Biology

Need to

Automate Sample

preparation

Need to

Automate Data Interpretation


Acknowledgements l.jpg
Acknowledgements Proteolysis-Mass Spectrometry

SGX

  • Julie A. Reynes, Michelle Buchanan and Chau Thai

  • Curtis Marsh and Boris Laubert

  • Ken Schwinn and Michael Sauder

  • Stephen K. Burley

  • Spencer Emtage

    Rockefeller University

  • Brian T. Chait and Martine Cadene

    Tecan

  • Brian Smith

    NIH NIGMS PSI


ad