Consultation and Project Design
We offer a free 45-minute consultation via Zoom video conferencing to all users. The core directors provide advice on usage of mass spectrometry and upfront assistance in experimental design of the projects. We consult users on suitability of methodologies, feasibility, detailed protocols and optimization routines, sample preparation, and evaluation of need for biological and technical replicates. Consultation and Project Design are included with all major project packages.
365 Proteome Profiling (Label-Free or TMT-Based)
Our “365” proteome profiling service allows identification and quantification of over 8,000 proteins from as little as 100,000 cells or 10-20 micrograms of tissue extract. These label-free or TMT-based profiling protocols are based on a dual-pH reverse phase fractionation of the total digested cellular proteome. The sample preparation method is coupled with sequencing on Thermo Scientific Orbitrap Eclipse, Lumos, or Exploris 480 mass spectrometers – the three most sensitive core instrument configurations. The data processing pipeline provides quantitation at MS1 level (gpGrouper approach). The standard data analysis includes recovery metrics, PCA analysis, hierarchical and k-means clustering, data normalization, differential expression (volcano plots), and GSEA pathway analysis.
Phosphoproteome & Proteome Profiling (TMT-Based)
The core offers combined phosphoproteome and proteome profiling based on a CPTAC TMT10/11 harmonized protocol. This profiling protocol includes extensive off-line basic pH reverse phase fractionation performed on Agilent Infinity 1260 HPLC system. A small amount of these fractions is measured directly for global proteome profiling, while the remaining fraction is processed for phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC). The sample preparation method is coupled with measurement on Thermo Scientific Orbitrap Eclipse, Lumos, or Exploris 480 mass spectrometers – the three most sensitive core instrument configurations. The data processing pipeline provides quantitation at MS1 level (gpGrouper approach). The standard data analysis includes recovery metrics, PCA analysis, hierarchical and k-means clustering, data normalization, differential expression (volcano plots), phosphosite level quantification and GSEA pathway analysis.
Affinity-Based Interactome Analysis
The core offers support for a variety of interaction studies based of protein enrichment/precipitation on an affinity matrix. These services include: 1. protein complex identification (protein-protein interactions via immunoprecipitation with primary or tag antibodies, or streptavidin pulldowns of BioID/TurboID/APEX assays), 2. protein-DNA interactions where DNA is used as a bait for protein binding (catTFRE transcription factor profiling, promoter pulldowns, response element pulldowns), 3. drug/small molecule target identification (binding to drug-bead conjugates), and 4. broad affinity pulldowns on commercial affinities (e.g., lectins).
Isolation and identification of protein interaction networks are our feature expertise. These services are based on knowledge accumulated by our personnel over multiple years of research (Jung SY, 2005, PMID: 16051665; and Malovannaya, 2010, PMID: 20133760, Malovannaya; 2011, PMID: 21620140). Using 1-20mg of total protein lysates, depending on the type of assay, we use a short affinity enrichment protocol, resolve protein complexes on SDS-PAGE, and analyze protein complex components on the Thermo Orbitrap Fusion mass spectrometer. The core offers options for antibody/affinity characterization, optimization of protein extraction, and cellular pre-fractionation in cases where interacting partners are sought for specific cellular compartments (e.g. nuclear or cytosolic). The staff is versatile in many variations of affinity-based protocols, including endogenous protein complex immunoprecipitations (IP/MS), affinity purification of tagged antigens (AP/MS), affinity enrichment (AE/MS), and cross-linked protocols (XL-IP/MS) for highest retention of transient interactors and membrane complexes. We provide comprehensive analysis of protein interaction results by filtering of non-specific identifications, annotating putative interacting partners, and mapping of interacting proteins onto reference protein complex networks as a standard part of our IP/MS project packages.
Single-Run LC-MS/MS Profiling
Our core performs routine measurements of proteins samples for protein identification and verification purposes. This service is generally reserved for simple samples such as, for example, a specific band of the SDS-PAGE gel-purified proteins. The single run profiling is also employed for low complexity samples - organelle preparations, extracellular matrix, or conditioned media.
Post-Translational Modification (PTM) Verification
Our core provides PTM (post-translational modification) analysis of affinity purified endogenous or ectopically expressed single recombinant proteins. PTMs that are readily analyzed include phosphorylation (on serine, threonine, and tyrosine), ubiquitinylation (on lysine), acetylation (on lysine), and methylation (on lysine or arginine). To identify modifications, raw data are searched against a modified protein database containing corresponding modification-specific mass changes. Trained core personnel manually validate all protein specific PTM spectral matches to validate site assignments.