Mass Spectrometry Proteomics Core Services

Instrumentation: Thermo Scientific Exploris 480 or Eclipse mass spectrometers

Our “365” proteome profiling service allows identification and quantification of over 10,000 proteins from as little as 100,000 cells or 10-20 micrograms of tissue extract. This TMT profiling protocol is 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 Exploris 480 or Eclipse mass spectrometers – the two most sensitive core instrument configurations. The data processing pipeline provides quantitation at MS1- level (iBAQ 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.

Instrumentation: Bruker timsTOF Ultra 2 mass spectrometer

The single run DIA based profiling service is offered for mid-coverage level and low complexity samples- biofluids, conditioned media, specialized population of cells, samples- organelle preparations, extracellular matrix, etc. The samples are measured on the current state-of-the-art Bruker timsTOF Ultra2 system which can identify over 6,500 proteins from a 20ng diluted Hela protein digest mixture.

Instrumentation: Thermo Scientific Exploris 480 or Eclipse mass spectrometers

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 portion 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 sequencing on Thermo Scientific Orbitrap Exploris 480 or Eclipse mass spectrometers – the two most sensitive core instrument configurations. The data processing pipeline provides quantitation at MS1- level (iBAQ 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.  

Instrumentation: Thermo Scientific Lumos or Fusion mass spectrometers

1. Protein Complex Identification

Isolation and identification of extended protein complex networks is a featured expertise of the Core. At heart of this service is the knowledge base accumulated by our personnel over the past 12 years of research (Jung SY, 2005, PMID: 16051665; and Malovannaya, 2010, PMID: 20133760). We have built custom algorithms to analyze IP/MS data and compiled a reference database of more than 5,000 endogenous protein complexes from ~4,000 IP/MS performed here at BCM (Malovannaya; 2011, PMID: 21620140). This is the most extensive endogenous human protein complex interaction dataset to date.

We start with ~5-20mg of total protein lysates, use a short affinity enrichment protocol, resolve protein complexes on SDS-PAGE, and analyze protein complex components on Thermo Orbitrap mass spectrometer. The core offers options for antibody 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 also 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 filtering of non-specific identifications, annotation of true interacting partners, and mapping of interacting proteins onto reference protein complex networks as a standard part of our IP/MS project packages.

2. Protein-DNA Interactions

3. Protein-Small Molecule Interactions 

Instrumentation: Thermo Scientific Lumos or Fusion mass spectrometers

Our core performs routine sequencing of proteins samples for protein identification and verification purposes. This singleshot DDA run is generally reserved for simple samples such as, for example, a specific band of the SDS-PAGE gel, recombinant proteins, PTM analysis on a particular protein, knock-out verification, etc. 

This service is mostly suited for targeted assays or cases that involve customization of sample processing pipeline or MS instrument method design. It includes systematic evaluation of best responders (peptides) for use in a Parallel Reaction Monitoring assay for proteins of interest as well as targeted analysis using Skyline software. 

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 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 address heightened false discovery rates that are inherent to the automated PTM matching algorithms.