The Center accepts any non-hazardous, non-pathogenic, and non-radioactive samples
for analyses. These samples can be whole organism (cell pellets, seedlings,
etc.), tissue (biopsies, roots, leaves, etc.), or purified protein (cell lysate,
precipitated protein, IPs, gel bands/spots).
We have standard operating procedures for each type of sample submission, but
we welcome discussion of the goals of the experiment prior to initiation of
a project (please contact Dr. Mooney - email@example.com).
- Two-dimensional electrophoresis
The Proteomics Center is equipped to perform high resolution two-dimensional
gel electrophoresis using the Amersham IPGphor or Bio-Rad Protean IEF units
and Ettan DALT II systems. The Proteomics Center will perform two-dimensional
electrophoresis on any non-hazardous biological samples provided by clients.
For highest probability of success samples should be free of salts and prepared
as dry precipitates (minimum of 0.1 mg protein) for direct resuspension in
isoelectric focusing media. However, the Center will accept whole cell or
tissue samples and prepare them for isoelectric focusing either by direct
solubilization in IEF media or by an alternative extraction
protocol developed by the Proteomics Center. It should be emphasized that
multiple samples should be prepared in the same manner if comparisons among
the gels are being made.
- Image analysis
The Center is capable of capturing images of Coomassie, silver, or fluorescent
stained gels in a 16-bit TIFF format. Currently gels can be analyzed in-house
using Phoretix 2D advanced image analysis software. Additionally, the Center
has a copy of the DeCyder program for analysis of DIGE (Cydye labelled) gels.
- In-gel digestion
of proteins for mass spectrometry
The Center will prepare tryptic peptide extracts from acrylamide gel sample(s)
excised on-site or brought in by the client. Non-trypsin protease digestion
can also be performed but must be discussed in advance with the Associate
- Mass spectrometry identification of proteins
Identification of proteins will be performed by obtaining masses and fragmentation
patterns of tryptic peptides and comparing these against in silico databases
using MASCOT, Sequest, and Spectrum Mill software. Non-complex samples (e.g.
2D gel spots) are identified using an Applied Biosystems 4700 MALDI TOF-TOF.
Increasingly complex samples are analyzed by liquid chromatography mass spec
(LC-MS) on a ThermoFisher LTQ Orbitrap XL or Agilent 6520 Accurate Mass QTOF.
- Intact mass analyses
Intact mass (molecular weight) of purified proteins are obtained using our
Voyager MALDI-TOF. Intact mass data have been acquired on proteins up to 150
kDa on this instrument. Additionally, an LCMS approach using a high-mass accuracy
instrument (Agilent 6520 QTOF which includes protocols specific for intact
mass analyses) can yield superior data (resolution and mass accuracy improvements)
for many samples.
- Post-translational modifications
The Center uses fluorescent stains and 2D gel electrophoresis to allow specific
imaging of phospho-proteins (ProQ-Diamond) and glyco-proteins (ProQ-Emerald).
For highest probability of success, mapping of post-translational modifications
by Mass Spectrometry should minimally begin with an enriched or purified preparation
of the modified peptide (or protein) and the deduced amino acid sequence of
the full-length polypeptide.
- Quantitative proteomics by mass spectrometry
LC-based separation of complex samples followed by simultaneous identification
of peptides/proteins is achieved using integrated accurate mass instruments
(LTQ Orbitrap XL or Agilent 6520 QTOF). We offer iTRAQ-based quantitation
and label-free (spectral counting) methods. The Center allows free client-access
to software for subsequent quantitative analyses (although Center staff can
also perform these analyses as fee-for-service).
- Training for software and instrument access
The Center emphasizes the full-service aspect of the core by involving staff
and investigator at all times during sample processing. We facilitate experimental
design, data analysis, manuscript figure/methods preparation, grant application
figures, methods, and facilities description preparation. Additionally, investigators
can be trained by staff on sample prep, software, and even instrumentation
if, for example, a project requires extensive mass spectrometric analyses.
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