The Proteomics Core facility is a collaborative research enterprise that provides state-of-the-art proteomics services to researchers from South Dakota and the surrounding region. Supported by the National Institute of General Medical Sciences grant and a partnership between the Sanford School of Medicine and the South Dakota Biomedical Research Infrastructure Network (SD BRIN), the core facility provides researchers with the capability to rapidly analyze and identify protein expression patterns in their experimental systems.
Along with providing proteomic analysis, the core:
- Develops experimental design, protocols, data analysis and interpretation
- Provides consulting and advice in grant proposal, as well as data preparation to be submitted to proteomics journal according to the requirements
- Offers training in the use of common equipment such as the scanner, spot cutter, imaging software, technique and protocol issues, and sample preparation.
This process identifies proteins from simple and complexes mixtures from different samples’ matrices using discovery proteomics through bottom-up approach, or peptide mass fingerprinting-MS or peptide fragment fingerprinting-MS/MS analysis.
The samples come in gel or in solution digestion of simple and complex mixtures (shotgun proteomics). Typically, protein spots or bands excised from 1D or 2D gels or from in solution are digested with trypsin and/or trypsin/Lys-C and the peptides resolved using a 1D or 2D NanoAcquity ultra performance liquid chromatography.
Tryptic peptides are desalted and concentrated using a reverse-phase trapping column and then resolved using a C18 reverse phase analytical column. The mass of peptides are determined using a nano-ESI Quadrupole-Time of Flight mass spectrometer in MS and MS/MS mode. The peptide masses are used to query various databases using several search engines, including ProteinLynx global server v3.0.3 Expression analysis, Mascot server and Proteome Discoverer to identify the most probable proteins in the sample.
We also provide:
- Protein characterization, including post-translational modifications and indirect protein-protein interaction analysis
- Protein quantification using label-free and labeled approaches
Peptide Mass Fingerprinting and Peptide Fragment Fingerprinting
We train in peptide mass fingerprinting and peptide fragment fingerprinting through in-gel or in-solution digestion of simple and complex mixtures. Typically, protein spots or bands excised from 1D or 2D gels or from in-solution are digested with trypsin and/or Trypsin/Lys-C and the peptides resolved using a 1D or 2D NanoAcquity ultra performance liquid chromatography.
Tryptic peptides are desalted and concentrated using a reverse-phase trapping column and then resolved using a C18 reverse phase analytical column. The mass of peptides are determined using a nano-ESI Quadrupole-Time of Flight mass spectrometer in MS and MS/MS mode. The peptide masses are used to query various databases, using ProteinLynx global server v3.0 Expression analysis or Mascot server to identify the most probable proteins in the sample.
Intact Protein Analysis
This type of analysis determines the molecular weight of an intact protein and can be used to identify post-translational modifications (phosphorylation, acetylation, etc.), proteolytic modifications and to determine the number of proteins in a sample. A typical procedure involves injecting the sample into the mass spectrometer using a regular electrospray or nano-electrospray configuration. Multiple charged spectra are generated and the spectra is deconvoluted using maximum entropy conversion software.
We provide training in using the Typhoon 9410 scanner, spot cutter, image software analysis and basic excision tools for the cutter.
We offer advice related to sample preparation, technique and protocol development or proteomics applications, as well as bioinformatics tools.
1D and 2D nanoAcquity Ultra Performance Liquid Chromatography
A liquid chromatography system that works in nano scale and allows you to separates small quantities of samples (fentomoles) using a nanoflow (200-400 nL/min) and also resolves complexes mixtures such as whole cell lysates, secretome and subcellular fractionation (nuclei and cytoplasm).
Ultimate RSLC3000 Ultra-High Performance Liquid Chromatography
A high-performance chromatography system with the capabilities to handle nano (50-1000 nL/min), capillary (1-10 ul/min) and micro flow settings (5-50 ul/min).
Synapt G1 HDMS
A hybrid Quadrupole-Time of Flight mass spectrometer with nano and regular spray ion sources and lockmass.
Orbitrap FTMS high resolution/mass accuracy (HR/MA) with enhanced resolution to 280,000 with a high-energy collision cell (HDC) with nano and regular spray ion source.
Typhoon 9410 Variable Mode Imager
A scanner with capabilities to work in fluorescence mode (four lasers and different filters), chemiluminescence and Phosphor imager (32P, 33P and 35S). Applications: Gels, pvdf membranes, well plates immunohistochemistry, in situ hybridization and micro arrays.
ProteomeWork Spot Cutter
A robot picker arm with a digital camera that allows to perform the automatic gel spots or bands cut either through visible or UV lights.
MassPrep Workstation Multiprobe II
An automatic liquid handling system programmable to perform different protocols such as gel processing, protein digestion and peptides extraction.
Solid Phase Extraction
A system with manifold and a dedicated vacuum pump to clean up and concentrate samples from different biological matrices.
Zoom IEF Fractionator
Provides a method to reduce sample complexity, enrich low abundance proteins and increase the dynamic range of detection. Solution phase isoelectric focusing with the ZOOM IEF Fractionator provides reproducible separations. Fractionated samples are ready for further analysis by two dimensional gel electrophoresis, one dimensional gel electrophoresis or two dimensional liquid chromatography/mass spectrometry.
SPD1010 Integrated SpeedVac
A centrifuge to dry and concentrate aqueous or non-aggressive samples with speed and cost-efficiency.
Used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the mobile phase) and a porous solid (the stationary phase).
Expedeon GELFREE 8100 Fractionation System
An electrophoresis system for MW-based fractionation with liquid phase recovery. The instrument is comprised of single-use, eight-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. The sample (proteins) can be loaded into a chamber, concentrated into a tight band in a stacking gel and separated based in electrophoretic mobilities in a resolving gel. Subsequently, the proteins eluted from the column are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This kind of system provides a simple and fast fractionation, is easy to use, and allows recovering of the proteins in liquid phase without the necessity to extract them from the gel. The system can fractionate a wide range of MW from 3.5-500 kDa and provides at least 4 different options of % of polyacrylamide. The GELFREE 8100 is a useful tool to be apply to the decreases of the complexity of the protein dynamic ranges present in complexes mixtures such as biological fluids, whole cell lysates and subcellular fractions.
|Name||Department & Office||Contact|
BASIC BIOMEDICAL SCIENCES
Lee Medicine & Science Hall 127
BASIC BIOMEDICAL SCIENCES
Lee Medicine & Science Hall 127
Lee Medical Sciences Building Room 127
414 East Clark Street
Vermillion SD 57069