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Midwest Proteome Center

MISSION

The Midwest Proteome Center is a new research resource facility at the Rosalind Franklin University of Medicine and Science, and comprises half of the Rosalind Franklin Structural Biology Laboratories.   As a facility in support of biomedical research in proteomics, we serve the ongoing research endeavors on campus and the scientific community at-large by providing state-of-the-art modern technologies at a reasonable cost.  The basic technologies employed include: protein separation, mass spectral analyses, protein identification, database mining and bioinformatics support.  Protein and proteome analyses are particular focal points of the center.

INTRODUCTION

A mass spectrometer, in simple terms, is a precise technology that measures the mass:charge ratio of chemical components of molecules.  This allows us to identify unknown proteins (proteomics) and metabolites (metabolomics) and characterize the complex of the non-covalent interaction between proteins and ligands OR proteins with other proteins.  As genomics, proteomics and metabolomics are pushing forward our understanding of life sciences, the analytical tools employed at the molecular level are becoming increasingly important.  Mass spectrometry has become an essential component underpinning some of the greatest discoveries of our times.

STATE-OF-THE-ART TECHNOLOGICAL CAPABILITIES:

PROTEIN/PEPTIDE SEPARATION:
UltiMate 3000 (Dionex) nano capillary LC separation system. Samples detected in femto-mol range. We implement the nano caplillary LC on-line or off-line coupling at the front-end of the QStar XL mass spectrometer in electrospray mode (see below) for high sensitivity and separation efficiency. The modules provide highest flexibility and address almost any front-end separation prior to MS.

 
2D GEL ELECTROPHORESIS:
DIGE [Differential In-Gel Electriophoresis]
DeCyder softwareFirst Dimension: 
LKB Multiphor Amersham Biosciences (GE Healthcare) IPGphor
Bio-Rad ROTOFOR- preparative  IEF cell 

Second Dimension:
Amersham Biosciences (GE Healthcare)Ettan DALT12

DETECTION:
Amersham Biosciences (GE Healthcare) Typhoon 9400

IMAGE RECOGNITION SOFTWARE
Phoretix 2D Evolution gel image analysis software:

Automated spot detection, matching and reliable image warping with flexible data display.
Batch process multiple analyses for high throughput
MW/pI determination using standards
Accurate densitometry and statistical evaluations
Cross-stain analysis for Differential Gel Electrophoresis (DIGE)
XML import/export of data

ROBOTIC SPOT PICKING:
Amersham Biosciences (GE Healthcare) Ettan Spot Picker

PROTEIN IDENTIFICATION
LC/MS/MS
We have the Applied Biosystems QSTAR® XL Hybrid LC/MS/MS System, a high-performance, hybrid quadrupole time-of-flight mass spectrometer designed for unknown proteins (proteomics) and metabolites (metabolomics).   As the highest-sensitivity hybrid quadrupole time-of-flight mass spectrometer, the QSTAR enables us to determine the mass and high-quality structural information of peptides, proteins and drug metabolites, as well as the type and location of post-translational modifications (PTMs).

MALDI-TOF

We have an Applied Biosystems Voyager-DE STR Biospectrometry
Workstation, a MALDI-TOF system (matrix-assisted laser desorption ionization-time-of-flight) that includes a 3-meter flight path, and improved ion optics with a high field detector.  It delivers high sensitivity, very good resolution and mass accuracy.  This instrument is used for the analysis of proteins, peptides, carbohydrates, etc.  In MALDI-TOF, the analyte is first co-crystallized with a UV-absorbing matrix, then submitted to pulse nitrogen UV laser radiation (337 nm).  This causes the vaporization of the analyte/matrix crystals and produces ions directed into a flight tube (time-of-flight).  The mass of an ion is measured by the time it requires to arrive at the detector (the smaller ions are faster, the larger are slower).  A total sample volume of up to 2 µl is loaded onto MALDI sample plate in volatile solvents.  The masses of peptides (low molecular weight, 750-4,500) can be determined on low fmol quantities with an average mass accuracy lower than 10 ppm.  Masses can potentially be obtained on numerous biopolymers that range from ~600 to >100,000 Daltons.

SELDI-TOF
We have acquired a Ciphergen Enterprise ProteinChip System Series 4000 for analyzing proteins captured on proprietary ProteinChip arrays in a high throughput format.   The Protein Chip Arrays provide a variety of surface chemistries for researchers to optimize protein capture and analysis.  The chemistries include chromatographic surfaces such as hydrophobic for reversed-phase capture, cation-and anion exchange surfaced for charged macromolecules, immobilized metal affinity capture (IMAC) for capturing metal-binding proteins, and pre-activated surfaces to investigate interactions such as antibody-antigen, receptor-ligand, etc.  A typical SELDI experiment is adding the protein sample, washing, adding the energy adsorbing molecule essential when analyzing a large number of samples to maximize reproducibility.  The chips are then processed in the mass reader where the bound proteins are liberated by ionization, and “fly” through a time-of-flight tube where   they separate based on mass and charge.  A bar code system is implemented for sample tracking. 

 

BIOINFORMATICS:

We are building a PLIMS (Proteomic Laboratory Information Management System).  Data is stored on a dedicated 2 Terabyte server and is mirrored for backups and archiving.  Gigabyte optic fiber wires the facility for data transmission between instruments and the investigator workstations throughout the facility.  Databases include public as well as proprietary sources.  Additionally, there are computational chemistry packages for structural proteomics initiatives in conjunction with our X-Ray Crystallography Facility across the hall.

MS Analyst, ProID, ProBLAST from ABI (instrumentation)

MASCOT Search Engine: A powerful database search engine on-line
Integrates all the proven methods of database searching
Peptide Fingerprint, Sequence Query and MS/MS Ion Search
Unique, true probability-based scoring
Total flexibility in chemical/post-translational modifications
Batch or Real-time mode

PRELIMINARY SAMPLE PREPARATION HINTS:

TYPICAL CONTAMINANTS IN PEPTIDE/PROTEIN SAMPLES

THERE IS NO OR LITTLE INTERFERENCE FROM:

formic acid, ßmercaptoethanol, dithiothreitol, very volatile organic solvents, HCl, NH4OH, acetic acid
TOLERABLE (<40 mM):
HEPES, TRIS, NH4Oac, octyl glucoside (<0.05%) TFA (<0.05%; a strong ion suppressor for ESI)
NOTE: minimizing bufferand solubilizing agents improves performance.  Use the minimum of these to control pH and solubility
AVOID:
glycerol, NaN3 (azide), DMSO, SDS, phosphate, NaCl, 1M urea, 1M guanidine, sucrose, TritonX-100 (<0.01%)

Helpful tips for sample preparation

Always use distilled and deionized water. Clean water is a critical condition for good sample preparation.
Keratin-like skeleton proteins are very common contaminants most of the time. They can come from protein extraction, gel preparation, or any step prior to trypsin. Always wear gloves and prevent any kind of contamination of keratin during sample preparation.
Sample clean up: using dialysis to cut off small molecules, ion, or cation exchange, reverse phase cartridges, Zip tips. These are available at cost from the facility.

SUPPORT:    Please acknowledge these grants in publications:
NIH NCRR S10 RR19325
HRSA C76 HF03610-01-00

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