Discovery Proteomics Service Line

IDeA National Resource for Proteomics Discovery Proteomics Service Line

The Discovery Proteomics Service Line specializes in discovery-phase proteomics. Discovery-phase proteomics refers to the large-scale identification and quantification of proteins or protein posttranslational modifications from a complex biological sample such as cells in culture, tissues, or plasma. High-resolution, rapid-sampling mass spectrometers operating in a data dependent mode (i.e., collecting data in real time) are the foundation of discovery-phase proteomics. Discovery phase proteomics generally utilizes 10s of samples and generates 1000s of candidate proteins for follow-up studies.

Sample Submission

The Discovery Proteomics Service Line 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. Projects will be assigned to this service line when the unbiased identification and quantification of thousands of proteins in each sample is required to meet project goals. The services described below are offered on a fee-for-service basis . For more information regarding sample submission or data analysis, please email Proteomics@uams.edu. A member of the lab will contact you to discuss facility usage in more detail.

For more information, see our iLab page.

Services

The sample preparation pipeline is organized to be robust and reproducible, as well as sufficiently adaptable so as to meet individual project requirements. Each of the specific sample preparation protocols routinely performed in the resource is described below.

Cell/Tissue Protein Extraction and Trypsin Digestion: The resource has protocols in place for efficient, high-throughput extraction of total protein from a wide variety of cell and tissue sample types by chloroform/methanol extraction or filter-assisted sample preparation. Both of these protocols integrate disulfide reduction, cysteine alkylation, and trypsin digestion into a single-day procedure.

Plasma/Serum Protein Depletion: Plasma and serum samples are prepared in a high-throughput manner for subsequent processing and analysis using HighSelect Top14 abundant protein depletion spin columns (Thermo Scientific) in order to reduce the dynamic range of protein concentrations in these samples. A variant of this protocol has also been developed by the resource for cerebro-spinal fluid (CSF) analysis.

Histone Extraction: Staff routinely isolate histone proteins from a wide range of biological samples by acid extraction for identification and quantification of epigenetic histone modifications, including acetylation, methylation, ubiquitylation, and phosphorylation.

SDS-PAGE and In-Gel Trypsin Digestion: The facility is equipped to run as many as eight SDS-PAGE mini-gels in a single day to prepare protein samples for gel-based analysis. The facility stocks 4-12% and 4-20% gradient gels to allow optimum resolution of different sample types. A high-throughput in-gel trypsin digestion protocol is also in place for rapid processing of gel bands or entire gel lanes.

Isobaric Labeling: The resource uses tandem mass tags (TMT; Thermo) to label tryptic peptides for quantitative multiplexing. Up to eleven isobaric TMT reagents are routinely used, each of which generates a unique quantifiable reporter ion during MS/MS or MS3 fragmentation of labeled peptide during a single analysis.

Phosphopeptide Enrichment: The resource has fully developed protocols for enrichment of phosphorylated peptides for analysis of the phospho-proteome. In addition to HighSelect TiO2 and Fe-NTA enrichment columns (Thermo Scientific) routinely used for general phospho-proteomics, a Src SH2 domain super-binder enrichment protocol is also being used for the selective enrichment and identification of tyrosine-phosphorylated peptides.

Peptide Fractionation: Peptides in complex samples are fractionated by reverse-phase chromatography on an Acquity BEH C18 column under basic pH conditions using a stand-alone UltiMate 3000 UHPLC systems (Thermo Scientific) prior to LC-MS/MS analysis under acidic pH conditions.

Custom service. Often investigators will require some hybrid of the above approaches or completely distinct approaches. These can be customized based on the needs of the user.

Instrumentation

The Discovery Proteomics Service Line is housed at UAMS and operates the below equipment:

Orbitrap Eclipse Mass Spectrometer

Orbitrap Fusion Lumos Mass Spectrometer

Orbitrap Fusion Tribrid Mass Spectrometers (2)

Orbitrap Exploris Mass Spectrometer

UltiMate 3000 UHPLC

Each Fusion-class mass spectrometer maintained at operated at UAMS is equipped withe the following options and accessories:

UPLC System Interface

Nanospray Ion Source

EASY-IC (internal calibration) Ion Source

Electron-Transfer Dissociation (ETD) Source 

Contact the UAMS site for any inquiries regarding instrumentation.