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The PMSF contains highly sophisticated
instrumentation that analyzes proteins (large biomolecules)
and metabolites (small biomolecules) contained in diverse
biological samples.
For biomolecule separations, we have several
gas chromatography,
liquid chromatography and
capillary electrophoresis chromatography systems that are off-line or
on-line with mass spectrometers. The liquid chromatography
systems provide excellent fractionation of mixtures of proteins
and peptides and facilitate downstream proteomic analyses.
A major component of protein separation work is carried out
with 2D gel electrophoresis. Here, the initial protein
separation is done through isoelectric focusing (IEF) using
the BioRad
Protean IEF Cell or
IPGphor from Amersham Biosciences. Proteins are then
separated by molecular mass using
Criterion/Criterion Dodeca Cell from BioRad or
Ettan Daltsix system from Amersham Biosciences. The
resulting 2D gels can be stained with Coomassie, Silver and
Sypro Ruby (all of which are compatible with mass
spectrometric analysis) or with more specific stains such as
ProQEmerald and ProQDiamond to identify glycosylated and
phosphorylated proteins, respectively.
High quality gel images are acquired with an
Epson Expression 1640XLGA scanner for Coomassie and
Silver stained gels, and a
Typhoon 9410 imager (Amersham Biosciences) for Sypro
stained gels. Gel images are then analyzed using
Progenesis SameSpots gel analysis software (Nonlinear
Dynamics Ltd.).
Following image analysis, interesting proteins are excised
from gels by
GelPix (Genetix Inc.) and digested by an automated
digester, the
MultiProbe II workstation (PerkinElmer Inc.).
For mass fingerprinting results, an aliquot
of the protein digest is zip tipped to remove salts and
spotted onto a MALDI target plate
and analyzed by a Voyager-DE STR
MALDI-TOF mass spectrometer (Applied Biosystems). The
acquired data is submitted to database searching and
protein identification using Protein Prospector (online
freeware).
If further mass spectral analysis is needed, the peptides
can be directly sequenced using the ESI (electrospray
ionization) source of the
QSTAR XL Q-TOF mass spectrometer (Applied Biosystems) or
separated by
nano-flow LC (LC Packings or
Eksigent NanoLC-2D) on the front end
of the ESI source of the MS/MS system. The acquired data is
submitted to database searching and protein identification
using MASCOT v2.2 database searching engine. We also have
additional bioinformatics tools for protein identification
and quantification using LC-based labeling techniques (ProteinPilot
2.0, Applied Biosystems) and for protein reporting (Scaffold
2.0, Proteome Software).
Additionally, we have a
4000 Q-TRAP LC/MS/MS system (Applied Biosystems), which
is a hybrid triple quadrupole/linear ion trap mass
spectrometer ideal for drug discovery and development,
metabolite identification and proteomic applications
including post-translationally modified proteins discovery
and biomarker validation. A
Shimadzu HPLC using an
auto sampler from Leap Technologies is coupled to the Q-TRAP
instrument for separation of complex samples and metabolite
quantification.
Automation of many of the steps in the above-described
process enables us to work in a high throughput and
efficient manner. Furthermore, we are also equipped with a
Waters
ACQUITY UPLC system for free
and hydrolyzed amino acid analysis.
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GCQ from ThermoFinnigan:
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HPLC System Gold from Beckman/Coulter:
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Autosampler
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Diode Array Detector
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UV/VIS Detector
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Fluorescent Detector
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Waters ACQUITY UPLC System provides:
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Reliable,
high quality chromatographic separations produced up to
10 times faster than current HPLC methods, reducing time
and cost per sample.
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Increased
efficiency for sharper peaks and better resolution,
resulting in better quantitative and qualitative
analyses.
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Low
dispersion, which yields up to a 5x increase in
sensitivity.
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A
variety of sample format options, from vials to plates,
with the capacity to automate and hold waiting samples
in a temperature-controlled environment.
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Used for AAA
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HPLC from Shimadzu:
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CTC PAL autosampler from
Leap Technologies
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UV detector
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Chromatography temperature
control
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Flexible system
configuration
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Nano-Flow HPLC system from
LC Packings:
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NanoLC-2D system from Eksigent:
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2-dimensional HPLC separations
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Pumping nanoscale HPLC system
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Eksigent's Microfluidic Flow Control (MFC) technology
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P/ACE MDQ Capillary electrophoresis system from
Beckman -
Capillary Thermoregulation with Recirculating Liquid Coolant -
Multi-format Sample Introduction -
High Sensitivity UV Detector -
Multiple Separation Modes -
Automatic Standards Co-Injection -
Large Volume Buffer Reservoirs
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Bio-Rad Protean IEF Cell and
Amersham IPGphor:
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First dimension isoelectric
focusing unit
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Take different immobilized
pH gradient (IPG) strips
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Built-in power supply and
cooling unit
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Criterion/Criterion Dodeca Cell
from BioRad:
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Second dimension gel
separation unit
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Runs 13.3 cm x 8.7 cm gels
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Large variety of precast
gels
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Two to four gel capacity
(Criterion Cell)
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Twelve gels simultaneously
(Dodeca Cell)
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PROTEAN II XL Cell:
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Ettan Dalt Unit from Amersham
Biosciences:
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Epson Expression Scanner:
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Typhoon 9410 imager from Amersham
Biosciences:
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High sensitivity and
resolution
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5-orders of magnitude
dynamic range
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Various imaging modes:
*PhosphorImaging
*Blue excited fluorescence
(457 nm and 488 nm)
*Green excited fluorescence 532 nm
*Red excite fluorescence 633 nm
*Chemiluminescence
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Used for SyproRuby gels and
DIGE gels
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GelPix spot cutting robot from
Genetix:
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8-channel excision head, 10
spots per minute
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Captures both visible and
fluorescent images
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Cooled CCD camera, 16-bit
Tif format
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15 x 96 well plates,
walkway automation
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MultiProbe II protein
digester from PerkinElmer:
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MALDI instrument:
Voyager-DE STR from
Applied Biosystems
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Positive and Negative
ion mode.
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Linear and reflectron
mode.
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PSD capable (Post Source
Decay).
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CID cell (Collision
induced dissociation).
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MALDI/ESI
Quadrupole Time-of-Flight:
QSTAR XL from
Applied Biosystems
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MALDI, Electrospray, and
Nanospray source.
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Ultimate LC unit for 1-
and 2D on-line chromatography
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Positive and Negative
ion mode.
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CID cell (Collision
induced dissociation).
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4000
Q-TRAP LC/MS/MS from Applied Biosystems:
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Enables
high-sensitivity full-scan MS,
MS/MS,
and MS3 with high-selectivity from true triple quadrupole
precursor ion (PI) and neutral loss (NL) scans.
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Perform multiple reaction monitoring (MRM)
for quantitation using high-sensitivity triple quadrupole.
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Identify, characterize, and quantitate
metabolites more quickly and easily.
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Identify and sequence post-translationally
modified (PTM) peptides automatically in a single LC/MS/MS
run.
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Facilitates hypothesis-driven
biomarker discovery and validation.
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Biacore 2000 from
Biacore Inc.:
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4 flow cells per chip allow two experiments including controls
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Autosampler with temperature control (4-40ºC)
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Affinity studies
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Kinetics
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Determination of valences
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Bound analytes can be eluted for subsequent MS analyses
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Progenesis SameSpot from NonLinear Dynamics
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Analyze single stain and 2D
DIGE experiments.
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Optimize analysis using
initial automatic image quality assessment and feedback
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Highly advanced and
automated image alignment.
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Create multiple image
groups to compare different views of data in the same
experiment.
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Automatic analysis of the
aligned image set and correction of dye related offsets
in DIGE
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Automatically create a
ranked list of significant spots based on ANOVA p-value,
fold-change and q-values for any groups you compare
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Confirm hits using 2D and
3D montage views, expression profiles and spot data
tables
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Use Principle Components
Analysis, Correlation Analysis and Power Analysis to
survey your data
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MASCOT Search Engine v. 2.2
from Matrix Science:
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A powerful database search
engine
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Integrates all the proven
methods of database searching
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Peptide Fingerprint,
Sequence Query and MS/MS Ion Search
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Unique, true probability-based scoring
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Total flexibility in
chemical/post-translational modifications
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Batch or Real-time mode
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Use any nucleic or amino
acid database in FASTA format
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Protein Pilot 2.0 from Applied Biosystems
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Protein identification and
relative protein expression analysis for discovery
research
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Optimized support for iTRAQ, ICAT, and SILAC reagent workflows, including the iTRAQ Reagent - 8plex.
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Enable to search for
hundreds of peptide modifications and non-tryptic
cleavages simultaneously
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Easily distinguish protein
subsets, and suppress false positives.
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Scaffold 2.0 from Proteome Software
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MS/MS analysis &
visualization software
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See proteins identified
across multiple samples.
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Combine Sequest and Mascot
scores for increased confidence.
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Get more detailed and
accurate information from your MS/MS experiments
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