Consultation and Project Design
The core directors provide advice on usage of mass spectrometry as a discovery technique 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 necessities for biological and technical replicates. Consultation and Project Design are included with all major project packages.
365 Proteome Profiling
Our “365” proteome profiling service allows identification and quantification of over 6,000 proteins from as little as 100,000 cells or 10-20 micrograms of tissue extract. This label-free 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 Orbitrap Fusion Tribrid or Thermo Scientific Q–Exactive mass spectrometers – the two most sensitive core instrument configurations. With such workflow, we can obtain an average of ~125,000 peptide spectra in 12 hours of sequencing time. These spectrometry data routinely identifies 6,000 proteins in each experiment. The sensitivity of the profiling methodology is suitable for the cost effective analysis of small scale or clinical samples. The label-free mass spectrometry-based protein quantification is standard with this service and can be effectively used for differential protein expression analysis.
Protein Complex Identification by IP/MS
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 ~20mg of total protein lysates, use a short affinity enrichment protocol, resolve protein complexes on SDS-PAGE, and analyze protein complex components on Thermo Scientific Elite 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.
Having established a protein complexome, 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.
Post-Translational Modification (PTM) Analysis
Our core provides PTM (post-translational modifications) 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), and acetylation (on lysine). 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.
Our core performs routine sequencing 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 raw spectral data are analyzed in Proteome Discoverer software (Thermo Scientific) with the Mascot search engine (Matrix Science). The assigned peptide spectra and peptide peak quantification are then processed through a gene-centric protein inference algorithm gpGrouper that accounts for distribution of shared peptide quantities (Saltzman/Malovannaya; 2018, PMID: 30093420).
To evaluate specificity of interacting partners, we rely on referencing our complexome study database and algorithms that were established by this work. The results are annotated and provided back as specific candidate lists and in align! data mining application or spreadsheet export. Quality control, normalization and primary analysis of differential expression and interactions are included as a package with the MS technology platforms. The Core can also perform batch correction, standard clustering analysis (hierarchical, K-means), PCA, volcano plots, and GSEA enrichment analyses as needed.
Secondary analyses are referred to the Bioinformatics Core. Examples of these data analyses include:
- Pathway mapping using commercial proteomics software (e.g. Ingenuity, GSA)
- Integration with orthogonal omics data (such as RPPA, transcriptomics, or metabolomics)