Proteomics & Metabolomics Grants and Publications

Proteomics & Metabolomics Shared Resource Grant Description

The Proteomics & Metabolomics shared resource provides support for mass spectrometric analysis including MALDI mass analysis, LC-ESI mass and tandem mass analysis. The proteomics facility is located at Fred Hutch’s South Lake Union Campus on the E level of the Thomas Building. Combined wet bench and instrument space is 2,100 sq. ft. Additional space is provided for proteomics data analysis computers and office space for resource personnel. Highly trained staff with over 80 years of combined mass spectrometry experience operate the shared resource’s instrumentation, provide sample separation services, carry out data analysis and provide consultation. 

Proteomics Services

LC-ESI-MS

Three liquid chromatography electrospray ionization, or LC ESI mass spectrometer systems are operated by the shared resource for proteomics analysis. The first is a ThermoScientific Vanquish Neo liquid chromatography instrument connected to a ThermoScientific Orbitrap Ascend w/FAIMS (field asymmetric ion mobility spectrometry) mass spectrometer. The second is a ThermoScientific nanoEasy1200 liquid chromatography instrument connected to an Orbitrap Eclipse w/FAIMS mass spectrometer. The third is a ThermoScientific nanoEasy 1000 liquid chromatography instrument connected to an Orbitrap Fusion w/ETD mass spectrometer. All of the systems are automated for use 24 hours per day, 7 days per week and the HPLCs are configured for flow rates of less than 500 nanoliters per minute and use columns that are less than 100 microns in internal diameter. The detection limit for peptides is less than 200 attomoles for all systems. These systems are primarily used for identifying proteins, characterizing post-translational modifications on proteins and peptides, global quantitative experiments (SILAC, iTRAQ, and TMT), and targeted quantification experiments (parallel reaction monitoring, or PRM).

Qualitative Analysis

Protein identification experiments are carried out on a variety of sample types, ranging from a single band observed on an SDS-PAGE gel to highly complex protein mixtures isolated from cellular lysates. Sample scales can range from sub-microgram up to hundreds of micrograms.

Quantitative Analysis

A number of common quantitative analyses are performed by the shared resource, including label-free quantification (area-under the curve), spectral counting, SILAC, iTRAQ and TMT. Highly sensitive and selective assays (targeted assays) are also performed using parallel reaction monitoring (PRM). Consultation with resource staff can help determine the appropriate analysis used for a sample type.

Protein Modification Analysis

Phosphorylation, acetylation, methylation and ubiquitylation are common modifications analyzed by the shared resource. Analyses range from peptide mapping experiment on a single protein to multiplexed, proteome-wide modification studies. The shared resource is especially experienced with qualitative and quantitative analysis of modifications on histones.

Sample Preparation

The shared resource provides expertise in preparing samples, primarily proteins and peptides for mass spectrometric analysis. Services include enzymatic digestion of proteins in solution or in gel slices and sample desalting and concentration. Additionally, the shared resource has extensive expertise in preparing samples, such as from serum or cell lysates, for multiplex quantitative analyses using iTRAQ and TMT isobaric chemical tags. HPLC purification is available for the purification and/or fractionation of protein and peptide mixtures via ion-exchange and reverse/basic-reverse phase separations. Isolation and enrichment of phosphopeptides via metal-based affinity techniques is also an available service. Consultation with the shared resource staff is encouraged for experimental design considerations, such as estimating protein quantities needed for analysis and identifying contaminants that might inhibit analysis.

HPLC

The HPLC instrumentation maintained by the core is made up of a Dynamax SD-200 preparative HPLC (milligram scale) with UV-Vis single wavelength detection, a Thermo Scientific Vanquish HPLC (1 µg to milligram scale) with UV-Vis multi-wavelength diode array detection, and a Michrom Paradigm HPLC (1 µg to 100 µg scale) with UV-Vis single wavelength detection. Each HPLC is equipped with a fraction collector that can collect into a variety of formats, including deep or regular well 96-well plates and 15 mL tubes. The Vanquish and Paradigm instruments are frequently used for basic reverse-phase fractionation of complex peptide mixtures and ion-exchange separations of globin chains from hemoglobin.  

Data Analysis

The shared resource uses ThermoScientific Proteome Discoverer v2.5 (PD 2.5) for analyzing data from virtually all LC-ESI data, including data from protein identification, quantification (including label-free, spectral counting, SILAC, TMT and iTRAQ data), and protein modification experiments. PD2.5 is operated on a OmicsPC Maximum Destroyer Ultra desktop computer equipped with 36 processors/72 threads for high-speed data analysis. The shared resource also uses the open-source software Skyline for the analysis of PRM and SRM data. Assistance with downstream data analysis (e.g. significance testing, pathway analysis, protein interaction analysis) is provided by the shared resource as well as the Hutch Data core. Data storage and archiving is maintained by the center’s Information Technology department. Data is distributed via a networked computer system providing all investigators immediate access to their data. A data management infrastructure is in place to handle data generated by the core. This system is scalable for expansion to meet future data storage needs.

Training

The shared resource periodically offers a week-long course on mass spectrometry-based proteomics. The course covers general principles of protein separation, mass spectrometry, and qualitative and quantitative proteomics. Data analysis is also covered and participants are introduced to the available data analysis tools.

 

Metabolomics Services

LC-ESI-MS: Two liquid chromatography electrospray ionization, or LC-ESI, mass spectrometry systems are operated by the core for metabolomics analyses. The first is a ThermoScientific Q-Exactive HFX mass spectrometer equipped with a ThermoScientific Vanquish HPLC. The second is a ThermoScientific TSQ Vantage connected in-line with a ThermoScientific Accela 600 HPLC and OpenAS autosampler. Both of these systems use heated electrospray ionization sources and they are configured for flowrates between 50 µL/min to 500 µL/min. The Q-Exactive HFX is primarily used for untargeted metabolite profiling experiments while the TSQ Vantage is used for targeted metabolite experiments.

Untargeted Metabolite Profiling

Three untargeted assays are currently offered by the shared resource. The first is the analysis of polar metabolites using HILIC, or hydrophilic interaction chromatography. This assay can identify approximately 200 known metabolites from biological samples, including cell culture extracts, tissue samples and plasma/media. Analytes are identified in positive and/or negative mode by retention time matching and accurate mass. The second assay is the analysis of phospholipids using C18 reverse-phase UHPLC. Based on accurate mass matching from positive and negative ionization modes, over 230 distinct phospholipid species can be detected. The third assay is for the analysis of saponified fatty acids. Using UHPLC C18-based chromatography with negative ionization, currently 20 fatty acids can be detected.

Targeted Metabolite Quantification

Using C18 reverse phase chromatography with SRM-based mass spectrometry detection, quantitative assays are available for adenosine, N⁶-methyladenosine, 5-hydroxymethyl-2’-deoxycitidine, and 5-methyl-2’-deoxycitidine.

Sample Preparation

The shared resource has experience with preparing samples from cell culture extracts, tissue samples and clinical samples for each assay type.

Data Analysis

Metabolite identification is made through retention time and accurate mass matching. For metabolite quantification, data are analyzed with Thermo Scientific Trace Finder 4.1 using a stand-alone desktop workstation. In addition, Thermo Scientific Compound Discoverer 3.0 is used in assisting with metabolite identification. The Fred Hutch Information Technology department handles all data storage, distribution, management and archiving functions through its networked systems.