Overview of Equipment and Services
The IDeA National Resource for Quantitative Proteomics provides a full range of services for protein characterization by mass spectrometry, including protein identification, mapping of post-translational modifications, global proteomics, and quantitative comparison of proteins in biological samples using state-of-the-art equipment. Our services are comprehensive, including complete sample preparation starting from cultured cells, frozen or FFPE tissue samples, serum or plasma, IP and other affinity purifications, as well as many other types of biological sample. Comprehensive data analysis is also included with all services. We are committed to working with each investigator to adapt our approach to fit specific research goals and to providing results in customized publication-quality form. Please contact us at IDeAproteomics@uams.edu to discuss how we can support your research.
IDeA Facility Equipment
Orbitrap Tribrid Mass Spectrometers
The Thermo Orbitrap Eclipse Tribrid system (Thermo Scientific) is the latest generation of the state-of-the-art Orbitrap mass spectrometry technology. The facility also operates an Orbitrap Lumos and two Orbitrap Fusion Tribrid instruments. 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 500,000 FWHM and 45 Hz scan rate. A linear ion trap performs collision-induced dissociation (CID) and electron-transfer dissociation (ETD).
Orbitrap Exploris 480 Mass Spectrometer
The Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) combines a quadrupole mass filter with a high-field Orbitrap mass analyzer and high-capacity ion transfer capability to optimize scan rate, sensitivity, and resolution. Improvements over earlier generation instruments in this class include significantly enhanced instrument sensitivity largely due to improved ion transfer capabilities, improved instrument robustness and serviceability, and compatibility with next-generation ion sources such as the FAIMS Pro (high field asymmetric ion mobility spectrometry) ion source. This instrument is primarily used in our facility for data-independent acquisition (DIA).
TSQ Quantiva Mass Spectrometer
The TSQ Quantiva (Thermo Scientific) is a triple quadrupole instrument used exclusively for selected reaction monitoring (SRM) experiments for targeted quantitative proteomics. The advantage for SRM experiments on the TSQ Quantiva is rapid switching between transitions with a set dwell time. The result is that a large number of peptides can be measured in a single run with a consistent 1.5 cycle per second acquisition rate, which gives at least 20 data points for all chromatographic peaks. Scheduling is used for all experiments based on the known retention times of targeted peptides. SRM on a triple quadrupole mass spectrometer remains a workhorse experiment for quantitative analysis with outstanding precision, lower limits of detection, and robust instrument operation.
Q Exactive Plus Mass Spectrometer
The Q Exactive Plus (Thermo Scientific) is a hybrid quadrupole-orbitrap instrument used for parallel reaction monitoring (PRM) in our targeted quantitative proteomics services. Orbitrap instruments are known for high m/z resolution, with values ranging from 17,500 to 280,000 on the Q Exactive Plus. The advantage for PRM experiments on the Q Exactive Plus is the high-resolution product ion m/z scan and the increased specificity of detection this m/z resolution provides via smaller m/z tolerances in the data analysis.
UltiMate 3000 Binary Analytical Liquid Chromatography System
The UltiMate 3000 UHPLC system (Thermo Scientific) is capable of precise and efficient offline fractionation of complex protein digests. This system is used in the facility 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.
High Performance Computing (HPC) Cluster
Our HPC is composed of 96 traditional Xeon CPU nodes each with 28 cores and 128 GB of memory (2688 CPU cores), 96 Xeon Phi nodes each with 64 cores (80 with 384 GB of memory and 16 with 192 GB of memory; 6144 Phi cores total), 4 Xeon nodes with 24 cores and 128 GB of memory plus 2 NVIDIA GPUs (96 CPU cores, 8 GPUs with 27,264 total cores), 6 management/login/storage interface nodes, and 1.9 PB of high speed storage (DDN Gridscaler). All components are interconnected via 100Gbps Omnipath interconnects and attached to external storage and the Internet (both commercial and I2) with redundant 10 Gbps Ethernet interfaces. Intermediate storage is provided by a 350 TB NAS and long term/backup storage is provided by a 4.2 PB object storage system (Dell/EMC ECS appliances).
We have two PowerStation GX2 Advanced Workstations (PSSC Labs). Each Linux workstation contains 40 Intel Xeon Scalable Processor Cores for a total of 80 cores with hyperthreading enabled, two GOLD 6148 processors, 128 GB of high performance memory, 22 TB of RAID5 storage, CentOS 64-bit operating system, and a maintenance kit from PSSC Labs.
Dell Windows PCs
The facility maintains ten Dell Precision workstations, each with solid state hard drives and 128 GB RAM, supporting a full range of data analysis software, including an in-house Mascot Server (Matrix Science), several MaxQuant (Max Planck Institute) installations, Scaffold Q+S and Scaffold DIA (Proteome Software), and PEAKS Studio (Bioinformatics Solutions).
IDeA Facility Services
Discovery Proteomics Services
Cell/Tissue Protein Extraction and Protease Digestion:
The facility 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 procedure. In addition, both protocols have been adapted to allow processing of large numbers of samples simultaneously.
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 resin (Thermo Scientific) in order to reduce the dynamic range of protein concentrations in these samples. Depletion of abundant proteins from these types of samples dramatically increases the number of lower-abundance proteins that can be identified and quantified by mass spectrometry.
Histone proteins can be isolated 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.
Tandem mass tags (TMT; Thermo Scientific) are used to label tryptic peptides for quantitative multiplexing. Up to sixteen isobaric TMT reagents are routinely used, each of which generates a unique quantifiable reporter ion during MS/MS or MS3 fragmentation of labeled peptides during a single analysis. Multiplexing of samples allows a several-fold increase in the efficient use of mass spectrometer time and improves data quality by eliminating variability resulting from analyzing samples from the same experiment at different times.
The facility has fully developed protocols for enrichment of phosphorylated peptides from a variety of sample types for analysis of the phospho-proteome. HighSelect TiO2 and Fe-NTA enrichment columns (Thermo Scientific) are routinely used for general phospho-proteomics, with analysis of both enriched and un-enriched samples to allow differences in protein abundance to be distinguished from changes in phosphorylation.
Peptides in complex samples are fractionated by reverse-phase chromatography under basic pH conditions using a stand-alone UltiMate 3000 UHPLC system (Thermo Scientific) prior to LC-MS/MS analysis under acidic pH conditions. The pH differential between the off-line and in-line separations results in an even distribution of peptides across up to 36 hours of mass spectrometer time, maximizing the number of identifiable peptides in each sample and allowing efficient use of instrument time.
The facility has implemented a DIA workflow harnessing the unparalleled performance of the Orbitrap Exploris mass spectrometer to acquire high-quality data for large sample sets. This approach is particularly well-suited to analysis of serum and plasma samples. DIA collects data for all peptides within defined mass-to-charge windows rather than detecting each peptide in a sample prior to fragmentation.
Targeted Proteomics Services
SRM and PRM Analysis
Selected reaction monitoring (SRM) analysis relies on the detection of unique transition signatures consisting of peptide-ion and fragment-ion pairs on a triple quadrupole instrument to quantify peptides and proteins across sample sets. Parallel reaction monitoring (PRM) analysis uses a quadrupole-Orbitrap instrument to perform a full scan of each fragmented peptide target. Each instrument has particular advantages for the targeted quantitative proteomics approach. The primary advantage of SRM experiments is rapid switching between transitions with aset dwell time. The primary advantages of PRM experiments are the added specificity of the analysis due to high resolution and the generally higher sensitivity of the Orbitrap. As a result, most projects will make use of both SRM for panels of high abundance proteins and PRM for panels of lower abundance proteins.
The advantage of SRM and PRM experiments described above is the high specificity of the analysis, but the experiments measure only the predetermined assay targets. High resolution accurate mass (HRAM) experiments are not biased and can be probed for any target peptide at any time, so the HRAM dataset is a valuable resource with the potential to measure large numbers of additional proteins without the need to repeat the initial biological experiment. The HRAM dataset also serves as a backup experiment that can enhance targeted SRM and PRM data with important results verified, if needed, by investigating additional peptides.
Targeted Assay Development
The facility currently has approximately 500 assays available for mouse and rat, with additional assays in human, yeast, fruit fly, and worm. We organize assays into panels of approximately 40 assays mostly based on different biochemical pathways. Commonly used panels include glycolysis, gluconeogenesis, Krebs cycle, mitochondrial and peroxisomal beta oxidation, proteostatsis, etc. In principle, a targeted assay can be created for any protein from any organism with a sequenced genome.
Full Data Analysis Support for Discovery and Targeted Services
The facility offers comprehensive data analysis with all proteomics services, including database searches, pathway analysis, statistical analysis, and preparation of interactive figures including heat maps and volcano plots. High-resolution, publication-quality figures are provided as needed for manuscripts and grant proposals. Further details can be found below.
Mascot and MaxQuant Database Searches
Proteins and peptides are identified and quantified by matching MS/MS spectra to database entries for any biological species with a sequenced genome. Post-translational modifications can also be included in search parameters. Quantitative results can be reported as spectral counts, peptide peak intensities, or TMT reporter ions.
We have developed in-house software for quality testing, filtering of peptides/proteins, testing various normalization methods, and performing differential analysis directly from database search results. proteiNorm has four major functions: (1) filtering options to remove outlier samples and low quality proteins/peptides, (2) normalization methods are tested quantitatively and qualitatively, (3) batch effects are identified and removed, (4) differential analysis is performed using the most appropriate statistical method.
To facilitate the process of interpreting statistical results form phosphor-proteomics studies, we have developed in-house software for exploration of processed data. Following data acquisition, ProteoViz performs data preprocessing, differential analysis, pathway and gene set enrichment analysis, identifies motifs and kinases, and provides visualization of all the results using an interactive dashboard. The protein analysis function can also be applied to label-free protein quantification.
For analysis of histone PTMs, we have developed in-house software that allows a user to upload histone PTM intensity values and perform quality control, normalization, differential expression, and visualization. An interactive dashboard allows users to filter samples and sample groups so they can visualize only a subset of data or plot a data distribution before and after normalization to evaluate different methods. The normalized data can then be visualized using interactive heatmaps, volcano plots, boxplots, and bar plots of histone PTMs.
Full bioinformatics support is provided for PRM, SRM, and targeted assays described in the sections above. Data are analyzed using Skyline and in-house R scripts, and peak areas for each transition are summed to generate quantitative intensity values for each sample. The calculated intensity values can then be loaded into the in-house software described above for visualization.
IDeA Facility Personnel
|Alan Tackett||Facility Directoremail@example.com|
|Mike Kinter||Facility Directorfirstname.lastname@example.org|
|Sam Mackintosh||Managing Directoremail@example.com|
|Rick Edmondson||Scientific Directorfirstname.lastname@example.org|
|Stephanie Byrum||Bioinformatics Directoremail@example.com|
|Aaron Storey||Research Scientistfirstname.lastname@example.org|
|Lisa Orr||Research Associateemail@example.com|
|Robert Brown||Research Technicianfirstname.lastname@example.org|
IDeA Facility Service Requests
Please contact us at IDeAproteomics@uams.edu to discuss how we can support your research.