Proteomics

Under the direction of Kumar Kolli, Ph.D., WRI's Proteomics is to discover the protein biomarkers associated with diseases such as Breast cancer, Gynecological cancers and Cardiovascular disease by generating the molecular profiles from healthy and diseased specimens using the state-of-the-art proteomic technologies. We have access to a wide variety of tissue and bodyfluid biospecimens which are procured from the consented subjects enrolled in a variety of protocols. Through the development of methods in the sample preparation and fractionation and utilizing advanced proteomic techniques such as 2D DIGE and LC/MS/MS, we are generating the proteomic patterns from the biological specimens that will allow to determine the global protein expression patterns and identify the proteins whose expressions are altered. The knowledge gained through the comprehensive proteomic analyses of clinical samples will aid in understanding the disease process and in the early detection of disease.
 

 2D gel electrophoresis

     This technology separates proteins based on their charge and size in two dimensions. We employ differential in gel electrophoresis (DIGE) in conjunction with 2D gel electrophoresis by labeling the proteome samples with cyanine dyes and pooling them together for simultaneous analysis in one gel. The cyanine labeled proteins are mixed with rehydration buffer and loaded onto the isoelectricfocussing (IEF) strip of a specific pH range. The IEF strip on which the proteins are focused will be loaded on to the SDS-PAGE for 2nd dimension separation of focused proteins.

     The gels will be scanned for protein spot profiles using a wavelength specific to the particular CyDye used. The CyDye images will be analyzed using DeCyder software and the differentially expressed spots will be picked and processed using a robotic spot handler workstation for MS analysis (Maldi MS and/or Nano LC/MS/MS). The MS data is interrogated with the protein databases by Profound and Mascot for protein identification.

     The internal standard which is one of three samples run on each 2D gel will compensate for experimental variations between the gels and allow us to normalize the spots across the samples (gels) for accurate protein expression analysis.

     An alternative to this approach a solution based two dimensional separations of proteomic samples is also in practice using a PF2D (two dimensional protein fractionation) platform for protein expression analysis. This technique separates proteins based on the charge and hydrophobicies respectively, in the two dimensions.

Multidimensional protein identification techonolgy (MudPIT)

     In this approach the proteomic samples are digested with trypsin and the peptide mixtures are separated on a two dimensional LC employing an ion-exchange and reversephase methods.

     2DLC is configured to separate the peptides based on charge and hydrophobicity in the first and 2nd dimension respectively and connected to an MS instrument (LCQ Deca XP plus, LTQFT) for online analysis of separated peptides. The peptide mixture is separated in several steps using a salt step gradient followed by reverse phase gradient. For each step a subset of peptides from SCX column are eluted onto a reverse phase column (C18) and the separated peptides on C18 column are mass analyzed and MS/MS analyzed by an MS instrument which operates in a data-dependant mode for tandem MS analysis. The MS is correlated to the protein databases for protein identification via the SeQuest and Mascot search engines.

     The development of label free quantitative proteomics methods using an lc/ms approach is underway on a newly acquired high performance LTQFT instrument.

     Offline fractionation of proteins is also performed on the PF2D in the first dimension before subjecting them to Mud PIT to increase the dynamic range and improve the detection of low abundant proteins.

Equipment and Data analysis tools utilized for proteomic analyses
 

• 2D Gel
  • Complete suite of GE Healthcare’s 2D DIGE equipment that comprise Ettan DALT 12 gel caster, IPGphor (two units), Ettan Dalt 6&II gel electrophoresis units, Ettan Spot Handler workstation and Typhoon 9400.

   

• Mass Spectrometers
  • Ettan Maldi Pro (GE Healthcare)
  • QTOF APIUS equipped with CapLC for online nano LC/MS/MS (Waters)
  • LCQ Deca XP Plus equipped with ProteomeX 2DLC (Thermo Electron) and Nanospray source
  • LTQFT equipped with ProteomeX 2DLC, MicroAS and Nanospray source

   

• LC instruments
  • Akta Explorer (GE Healthcare)
  • PF2D (Beckman)

   

• Software tools (Data analysis)
  • Decyder 5.02 (GE Healthcare)
  • BioWorks 3.2 (Thermoelectron)
  • Mascot 2.0 (Matrix-Science)
  • Profound (Proteometrics)

Proteomics Staff:

Kumar Kolli, PhD	Senior Director	k.kolli@wriwindber.org	814 - 361 - 6925
David Kirchner	Research Associate III		814 - 361 - 6957
Ric Katenhusen	Research Associate II		814 - 361 - 6956
Bernie Seth	Research Associate II		814 - 361 - 6975
Luke Weaver	Research Associate I		814 - 361 - 6918