Automated Edman chemistry is performed on an Applied Biosystems 492 Procise Protein Sequencer.  The Procise is a complete, automated chemistry and analytical system for amino acid sequence analysis of protein and peptide samples in picomole to nanomole amounts.

The Procise 492 Protein Sequencer performs automated Edman sequencing chemistry on a Biobrene-coated Glass Fiber Filter (for liquid samples) or on proteins blotted to PVDF after PAGE separation. Sensitivity is in the low pmol range.

 

 

 

Amino acid analysis is performed on a Hewlett Packard AminoQuant II system.  The system consists of a Hewlett Packard 1090 liquid chromatograph that is equipped with a programmable autosampler for automated sample handling, derivatization and injection, a diode array detector for low-moderate sensitivity (ultraviolet detection) analyses and an in-line Hewlett Packard 2400 fluorescence detector for high-sensitivity analyses (fluorescent detection).  All system control and data analysis is performed by a Hewlett Packard 2-Dimensional Chemstation.  Liquid and vapor phase hydrolysis protocols have been developed in order to service a wide range of sample types (liquid, dried and electroblotted proteins on membranes).  We offer routine validated (qualitative and quantitative) amino acid compositions of purified proteins and synthetic peptides as well as (quantitative ) free-amino acid (or total amino acid) compositions of feeds, flours and physiological fluids.  Hydrolyzed proteins samples and free amino acids are derivatized pre-column with o-phthalaldehyde (OPA) and 9-fluoromethyl-chloroformate (FMOC) prior to separation and quantitation by reverse phase HPLC.  Sensitivity is in the picomole to nanomole range depending upon sample matrix contributions.

 

 

 

Electrophoresis has been a valuable method of protein analysis for decades.  In recent years, the increased sensitivity of automated sequencers and analyzers has enabled the use of electrophoretic separation techniques to be used as preparative methods for subsequent stages of high-sensitivity protein analysis.  The Laboratory is fully equipped to perform a variety of routine electrophoretic separations of proteins in polyacrylamide or agarose matrices. Sodium dodecyl sulfate polyacrylamide (SDS) and non-denaturing gels are available in standard and mini-gel format (linear and gradient format) as are 2-dimensional electrophoretic separations. Analytical isoelectric focusing (IEF) is performed on an Amersham Pharmacia Biotec IPGPhor electrophoresis module (first dimension) using immobilized pH gradients in polyacrylamide strips (DryStrips).  The second dimension of resolution is obtained on a vertical slab gel electrophoresis unit equipped with an external re-circulating cooling device.

 

Electroblotting of proteins to solid membrane supports has been used for many years in protocols used for immunodetection of antigens.  Recent developments in membrane support technology have led to the production of high-capacity, chemically inert matrices that are resistant to N-terminal sequencing chemistries.  Proteins blotted to these matrices can be excised from a mixture of proteins and subjected directly to sequence analysis or preliminary manipulations such as direct In situ chemical or proteolytic cleavage with subsequent peptide extraction and purification.  The Laboratory is equipped to perform electroblotting procedures as well as In situ  or "in gel" digestion procedures as required to obtain sequence information and protein identity.

 

 

Typhoon Trio Fluorescent Imager

The Typhoon Trio (GE Healthcare) is a high performance gel and blot imager. The imager has powerful laser excitation sources and high-quality confocal optics that allow for the sensitive detection of low-abundance targets. Red, Green and Blue excitation wavelengths (488 nm, 532 nm, 633 nm) and a wide choice of emission filters enable imaging of an extensive variety of fluorophores. The Trio is also useful for Storage Phosphor Autoradiography, Chemiluminescence and Chemifluorescence detection. (Client must have their own Phosphor screens. The PCL does not own any.) Please call ahead with specific questions. The instrument is used on a first-come, first-served basis, but reservations are recommended for analyses that will require extensive periods of time.

The Digester is a highly versatile instrument designed to perform in-gel digestion of proteins captured in 2D gel electrophoresis spots. Default digestion protocols can be customized. The instrument performs automatic tracking of samples.

 

The Ettan Spotter is designed to automatically mix and spot digested proteins and matrix onto any MALDI sample plate.

 

High Pressure Liquid Chromatography 

Liquid chromatography is used extensively to purify proteins and peptides from mixtures of natural materials.  The PCL has a stand-alone Hewlett Packard 1100 HPLC system is equipped with an autosampler and a diode array detector (DAD) for spectral analysis and is used for narrowbore, microbore and capillary LC peptide mapping and purification using reverse phase chromatographic techniques.  This instrument is used in conjunction with the MALDI-TOF and Hewlett Packard G1005A Protein Sequencer to obtain internal protein sequence information from protein digests that can allow investigators to identify their proteins of interest and aid in the design of the appropriate probes for their isolation and, possible, cloning.  An LC Packings Probot is attached to the HP1100 HPLC to aid in automated spotting of peptides to a sample plate for MALDI-TOF mass spectrometry.  In addition, the facility has a Pharmacia Acta Explorer 10 Protein Purification and a GE Healthcare AKTA Purifier system that are capable of running aqueous and organic mobile phases for native protein purification as well as peptide isolation.  These instruments are being used to develop pilot protein purification protocols using a library of columns that includes hydrophobic interaction, ion exchange, metal chelation and affinity matrices.  In addition, the systems are used for Immuno-Depletion of contaminating proteins from physiological samples prior to Proteomic analysis.

 

 

MALDI-TOF Mass Spectrometer

 

The PCL has a Shimadzu/Kratos MALDI-TOF mass spectrometer equipped with delayed extraction and reflectron capabilities which is used for routine high mass accuracy analysis of peptides (<3 kDa). The instrument can also be operated in linear mode for protein analysis (>3 kDa).

 

 Electrospray Mass Spectrometer

We are equipped with a ThermoFisher DecaXP ion trap mass spectrometer that is used for peptide mass measurements and fragmentation (de novo sequencing).  Samples can be applied by direct infusion or after in-line reversed phase chromatographic separation.  Automated search algorithms (Sequest and MASCOT) are available to perform database searches to aid in protein identification.  This instrument is for peptide/protein analyses only.  If you are interested in small ion mass spec analysis, we recommend that you contact the Laboratory for Biological Mass Spectrometry in the Department of Chemistry at Texas A&M Unversity.