The 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. We offer the services described below on a fee-for-service basis to both University of Arkansas for Medical Sciences (UAMS) and outside investigators.

Additional Information
Services Provided
Contact Information

Overview of Core Services

Services Provided

Protein Identification
We offer in-gel trypsin digestion and tandem mass spectrometry (MS/MS) analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein bands. This service includes Mascot database searching. Protein identification 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 mass spectrometry (MS)-compatible. Silver staining is not recommended. Any visible gel band contains sufficient protein for identification. This service has a success rate of over 99.9 percent.

Mapping of Post-Translational Modifications
Core instrument systems are capable of detecting most stable post-translational modifications (PTM), 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
In-gel digestion followed by liquid chromatography (GeLC) is a high-throughput application of the protein identification method described above. In GeLC, an entire SDS-PAGE 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 MS. 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.

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

  • ICAT (Isotope-Coded Affinity Tags) is a quantitative isotopic labeling method. In ICAT, proteins or peptides from two different biological samples are labeled with chemically identical, isotopically distinct tags (e.g., a D0- vs. D6 thiol- reactive label). Labeled samples are then mixed and analyzed by MS. Peptides from different biological samples can be distinguished and quantitatively compared based on their distinct isotopic tags.
  • iTRAQ (isobaric tags for relative and absolute quantitation) is a quantitative peptide labeling method. In iTRAQ, 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 MS. The specific advantage of iTRAQ is that all labeled peptides are initially of equal mass and chemically identical. During MS/MS fragmentation, peptides from different biological samples yield distinct low-mass reporter ions that can be quantitatively compared.
  • SILAC (stable isotope labeling by/with amino acids in cell culture) 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 MS. Peptides from different biological samples can be distinguished and quantitatively compared based on their distinct isotopic labeling patterns.
  • I-DIRT (isotopic determination of interactions as random or targeted) is a variation of SILAC in which co-cryogenic lysis and immunopurification are used to distinguish specific protein interactions from nonspecific contaminant interactions. Subsequent MS analysis distinguishes specific (pre-lysis) interaction from post-lysis contamination based on the ratio of heavy/light isotope present. This method is particularly useful for characterization of multi-protein complexes.
  • 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 MS applications.


Contact Information

Proteomics Core
Biomedical Research Building I, Room B424A
4301 West Markham Street
Mail Slot 516
Little Rock, AR 72205

Ricky Edmondson, Ph.D., Co-Director
Myeloma Institute for Research and Therapy
UAMS College of Medicine

Alan Tackett, Ph.D., Co-director
Department of Biochemistry and Molecular Biology
UAMS College of Medicine

Samuel Mackintosh, Ph.D.
Department of Biochemistry and Molecular Biology
UAMS College of Medicine

Linley Moreland, M.S.
Department of Biochemistry and Molecular Biology
UAMS College of Medicine