Mass Spectrometry Core

Protein extraction and digestion from samples from various matrices are provided including experimental model cells, clinical samples (formalin-fixed paraffin-embedded, fresh frozen tissue, serum/plasma). In gel digestion can be performed to analyze a band or region of interest from a gel. In addition, the Proteomics Core provides recommendations for stable isotope labeling with amino acids in cell culture (SILAC) or performs tandem mass tag (TMT) labeling post digestion to allow for sample multiplexing. Other services include enrichments for post-translational modifications such as phosphorylation, ubiquitylation and immune- and affinity purification of protein fusions to define protein-protein interactions and proximity relationships. Offline fractionation is also offered to yield increased sequencing depth and coverage of the proteome.

Untargeted proteomics studies can identify >5,000 protein groups from an unfractionated sample and >9,000 protein groups from a fractionated sample. Protein identification and relative quantitation between samples can be determined (label free, SILAC, TMT). The data processing and statistical analysis are performed with MaxQuant or Proteome Discoverer, including database searching, retention time alignment, quantitation, and filtering.

Targeted proteomics analysis in hypothesis-driven experiments involves obtaining quantitative data on a predefined set of peptides. Core staff are available to help with stable isotope labeled (SIL) peptide library design, ordering, and optimization. In addition, access to current MTAC peptide libraries is available to all DRC users. Depending on the size of the library and the goal of the project, established techniques include parallel reaction monitoring (PRM), SureQuantTM, and absolute quantitation using validated assays.

Untargeted metabolomics studies are applied as a hypothesis generation strategy. Simultaneous qualitative and quantitative measurement of large number of detectable metabolites in samples has the potential to identify novel signatures.

We quantify low molecular weight organic biomolecules including fatty acids and related molecules; small organics; amino acids; carbohydrates; sterols, isoprenoids and polyols; glycerolipids; cardiolipins; sphingolipids; nucleosides and nucleotides; oxidative stress metabolites; and drug and natural product metabolites. In all, the core has expertise in routine characterization and quantification of >1400 small molecule metabolites (Table 1).

Targeted Metabolites Routinely Analyzed by DRC Metabolomics Core

The Core assists investigators in identifying unknown molecules from biological extracts by MS methods (ESI/MS/MSn) and interpreting mass spectra

The Core provides access to validated assays required for quantitative analysis of large sample sets from diabetes-related clinical studies and trials. We also develop FDA-compliant validated assays needed for measurement of analytes for clinical trials.

Flux analysis is performed in vitro or in vivo using stable isotope-labeled tracers and LC-MS/MS to monitor isoptologue composition of targeted metabolites and enable tracking of metabolic flux through pathways.

The Core instructs DRC members and their laboratory personnel in the principles of various modes of MS and their applicability to analyses of biological samples and in the preparation of samples for MS analyses. Because MS analyses require specialized skills, it is more economical and practical for MSCL/Metabolomics/MTAC staff to perform most analyses, but members of users’ laboratories are trained to perform some procedures, particularly if requirements are large and sustained.