UAMS Proteomics Core (proteomics@uams.edu)

Overview of Services

The UAMS Proteomics Core provides the service of protein characterization by mass spectrometry. This includes identification of unknown proteins, quantitative comparison of proteins in biological samples, and mapping of post-translational protein modifications. Please contact core personnel at proteomics@uams.edu to discuss how the core can support your research.

Proteomics Core Equipment

Orbitrap Fusion Mass Spectrometers. The Thermo Orbitrap Fusion Lumos system is the latest generation of the state-of-the-art Orbitrap mass spectrometry technology installed at UAMS in July 2017. The Proteomics Core also operates two Orbitrap Fusion Tribrid instrument installed in 2015. The Orbitrap Fusion systems combine a quadrupole mass filter with Orbitrap and linear ion trap mass analyzers in a novel configuration that maximizes ion transfer efficiency, allowing flexible combinations of different fragmentation techniques, scanning modes, and mass analyzers. The quadrupole mass filter allows precursor ion selection, enabling parallel use of the mass analyzers and synchronous precursor selection for MS3 analysis. An ion-routing multipole serves as a higher-energy collisional dissociation (HCD) cell and also transfers ions efficiently between the Orbitrap and linear ion trap mass analyzers, allowing use of either mass analyzer in combination with any ion fragmentation technique. The ultra-high-field Orbitrap mass analyzer has a resolving power of up to 450,000 FWHM and 15 Hz scan rate. A linear ion trap performs collision-induced dissociation (CID) and electron-transfer dissociation (ETD).

Orbitrap Velos Mass Spectrometer. The Thermo LTQ Orbitrap Velos is an earlier generation of the Orbitrap technology that has been maintained and operated by the Proteomics Core since 2011. This instrument system combines the rapid data acquisition capability of the LTQ linear ion trap with the high resolution of the Orbitrap. This configuration allows extremely rapid in-depth proteomic analysis at high mass accuracy and supports quantitative isotope-based proteomic profiling. The Velos configuration is also capable of HCD and ETD complementary to CID, which is available with earlier LTQ-based instruments.

nanoAcquity Ultra Performance Liquid Chromatography (UPLC). Each of the mass spectrometers described above is interfaced with a Waters nanoAcquity UPLC system, including a binary solvent manager and a sample manager. Precise chromatography is achieved by these systems through low detector cell volume, minimized tubing volumes and specialized fittings. The binary solvent manager and injector can sustain pressures up to 15,000 psi and can generate high-pressure gradients at nanoliter flow rates with minimal delay. The sample manager can accommodate two 96-well plates in a microtiter plate format.

UltiMate 3000 Binary Analytical LC System. The Thermo UltiMate 3000 System is capable of precise and efficient offline fractionation of complex protein digests. This system is used in the Proteomics Core workflow to fractionate peptides at basic pH in order to complement the acidic pH separation performed during liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. The UltiMate 3000 can sustain pressures as high as 9000 psi and flow rates as low as 50 nL/min. The system includes an analytical autosampler and fraction collector to allow efficient processing of large numbers of samples, as well as a UV detector to monitor peptide elution and prevent cross-contamination.

Computers and Software. The core maintains eight Dell Precision workstations, each with solid state hard drives and 128 GB RAM, running an in-house Mascot Server (Matrix Science), several MaxQuant (Max Planck Institute) installations, Scaffold Q+S, PTM and perSPECtives (Proteome Software), and PEAKS Studio (Bioinformatics Solutions). The core has 150 TB of dedicated data storage space to maintain secure backups of all data files.


Proteomics Core Services

Protein Identification
We offer in-gel trypsin digestion and MS/MS analysis of SDS-PAGE protein bands. This service includes Mascot database searching. Protein ID and peptide sequence information are provided to the user as a Scaffold data file. No special sample preparation or gel protocols are required. Coomassie and SYPRO staining are both MS-compatible. Silver staining is not recommended. Any visible gel band contains sufficient protein for identification.

Mapping of Post-Translational Modifications
Core instrument systems are capable of detecting most stable post-translational protein modifications, as well as identifying sites of modification. PTM detection is limited by stoichiometry of modification, ionization efficiency of modified peptides, and PTM stability. Enrichment of the protein or PTM of interest is typically necessary in order to achieve a high likelihood of success. Please consult with core personnel regarding the best approach for your specific interests.

GeLC Analysis
GeLC is a high-throughput application of the in-gel protein identification method described above. In GeLC, an entire SDS-PAGE gel lane is gridded into 2-3 mm slices, each of which is then subjected to trypsin digestion and MS/MS analysis. The principal advantage of GeLC is that it does not require that a protein be detected by gel staining prior to identification by mass spectrometry. Every protein present in the gel lane at or above the MS sensitivity threshold can potentially be detected. Since state-of-the-art mass spectrometers are several orders of magnitude more sensitive than the best gel staining methods, GeLC is a much more powerful method than detection/identification of individual SDS-PAGE gel bands or 2D gel spots.

Basic pH UHPLC Peptide Separation
This technique offers an alternative to the GeLC approach described above. bHPLC separation is able to achieve similar or better proteomic depth without running samples on SDS-PAGE. Instead, following trypsin digestion, peptides are separated into 6-12 fractions on a Thermo UltiMate 3000 UHPLC. Each fraction is then analyzed by mass spectrometry. With the Obitrap Fusion instrument, this technique can identify 10,000+ proteins in a single sample.

Quantitative Analysis
Orbitrap mass spectrometers are capable of mass measurements accurate to 5 ppm or better and are therefore ideal for quantitative mass spectrometric applications. Some common applications are described below. Please contact core personnel to discuss the best approach for your specific interests.

TMT is an isobaric quantitative peptide labeling method. In TMT, proteins from two or more biological samples are digested, and the resulting peptide products from each sample are labeled with a different isobaric tag. Labeled samples are then mixed and analyzed by mass spectrometry. The specific advantage of TMT is that all labeled peptides are initially of equal mass and chemically identical. During MS/MS and MS3 fragmentation, peptides from different biological samples yield distinct low-mass reporter ions that can be quantitatively compared.

SILAC is a method for metabolically labeling and quantitatively comparing proteins from different biological samples. In SILAC, cells are cultured in the presence or absence of one or more isotopically labeled amino acids. The cells are then harvested, proteins isolated and digested, and the resulting peptide products are mixed and analyzed by mass spectrometry. Peptides from different biological samples can be distinguished and quantitatively compared based on their distinct isotopic labeling patterns.

Label-Free Quantification is a method by which discriminatory unlabeled peptides are used to make quantitative proteomic comparisons between different biological samples. It has the obvious advantage of not requiring chemical or metabolic labeling, but is far more demanding in terms of planning, execution, and data analysis than other mass spectrometric applications.

Location and hours of operation

Hours Location

Facility and staff hours are     
by appointment only  

W.P. Rockefeller Cancer Institute, Rm 9149
4301 W Markham St
Little Rock, AR 72205

Shipping Address:
UAMS Biomedical Research Center I, Rm B421
325 Jack Stephens Dr
Little Rock, AR 72205

Contacts

Name Role Phone Email Location
Alan Tackett
Director
 
(501) 686-8152
 
ajtackett@uams.edu
 
Biomed I, B405E
 
Rick Edmondson
Director
 
(501) 766-3665
 
rdedmondson@uams.edu
 
WPRCI, 9121
 
Sam Mackintosh
Assistant Director
 
(501) 526-6165
 
sgmackintosh@uams.edu
 
WPRCI, 9111
 
Lisa Orr
Research Assistant
 
(501) 686-5954
 
orrlisam@uams.edu
 
Biomed I, B406
 
Renny Lan
Data Analyst
 
(501) 526-5238
 
slan@uams.edu
 
WPRCI, 9110
 

Services

Name Description