Mass Spectrometry-Based Differential Proteomics
Mass spectrometry-based differential proteomics is a comprehensive analysis of protein expression that involves comparing distinct proteomes, such as cells, tissues or cell lines that are normal, diseased or treated.
This type of analysis works best for defining differences between groups or treatments and for the initial "discovery" of potential biomarkers. For this service, the Proteomics Core currently employs the label-free approach and the stable isotope labeling by amino acids in cell culture (SILAC) approach.
The label-free approach uses one sample per gel lane and works on any type of material — assuming sufficient protein concentrations — from which proteins can be extracted.
The SILAC approach, most commonly done with cell lines, uses labeled media to metabolically incorporate an isotopic-labeled amino acid — lysine, arginine or both — into all proteins within the cell culture. Proteins from the SILAC culture are equally mixed with proteins from a corresponding unlabeled culture, with unlabeled-to-labeled protein abundance reported.
Considerations. Good experimental design is crucial. Careful selection of the number of samples and how they are acquired, stabilized and stored is critical for success. Protein extraction of each biological or technical replicate must be done in the same manner to assure proper comparisons. Accurate total protein quantitation needs to be done on all samples for equal sample mixing if doing SILAC and equal gel loading.
The wealth of information acquired can be overwhelming, so clients should have a plan on how this information will be utilized downstream — for example, it can be used for pathway analysis or for comparison with genetic data. There should also be a plan to corroborate mass spectrometry data with data from orthogonal methods.
Process. Protein preparations, done in replicates, are brought to the Proteomics Core to run 1-D SDS-polyacrylamide gel electrophoresis (1-D SDS-PAGE) gels followed by staining with colloidal blue. As an alternative, the gel can be provided by the investigator.
Each gel lane is divided into sections. The gel is cut accordingly, each gel piece is trypsin-digested, data is acquired with a Thermo Scientific LTQ Orbitrap mass spectrometer and comparative data analysis is done using Rosetta Elucidator software.
- Reporting. A typical report includes a list of proteins for each gel section with each protein's relative abundance change of one replicate group versus another replicate group — or unlabeled group versus labeled group — along with an estimate of the statistical likelihood of random results (a P value).
Prospective users should contact the Proteomics Core to obtain a project request form. The completed form will be reviewed by core staff and a follow-up meeting will be scheduled.
Before project approval, it is important to ensure that everyone involved has a complete and clear understanding of the project. The core also encourages users to consult with a biostatistician for guidance on both study design and data analysis.
The cost of a differential proteomics project can vary greatly depending on sample size, number of agreed-upon gel sections and analysis difficulty. A cost estimate is provided to clients prior to starting a project.
Read more about the label-free and SILAC approaches:
For more information or to initiate a service or project request, email the Proteomics Core.