The use of mass spectrometry for the characterization of individual or complex protein samples continues to be one of the fastest growing fields in the life science market.
Bottom-up proteomics is the traditional approach to address these questions. Optimization of each the individual steps (e.g. sample prep, digestion and instrument performance) is critical to the overall success of the entire experiment.
To address issues that may arise in your experimental design, Promega has developed unique tools and complementary webinars to help you along the way.
Here you can find a summary of individual webinars for the following topics:
Optimization of sample prep and digestion
Common shortcomings of protein mass spec sample preparation include incomplete digestion, lengthy and laborious procedure, low protein sequence coverage; and inefficient peptide recovery of in-gel digested proteins. This webinar will discuss the advantages of trypsin preparations with enhanced proteolytic efficiency. The webinar will also review mass spec—compatible SDS surfactant analogs and their advantage for in-gel protein digestion and peptide recovery.
While trypsin is an excellent protease, alternative proteases are useful for numerous applications including increasing protein sequence coverage and identifying post-translational modifications.
rAsp-N is a recombinant protease that displays both high cleavage efficiency and a strong preference for cleavage N-terminal to aspartic acid that will digest a variety of sample types. In addition, a multi-enzyme approach can be successfully employed to achieve 100% sequence coverage of an IgG from a single injection.
Preparation of protein samples for mass spec analysis is often the first of several bottlenecks in bottom-up proteomics workflow. The proteolysis step is often carried out overnight, extending mass spec analysis to 2 days. We have developed a modified protease mix of trypsin/lys-C and an optimized digestion buffer to shorten proteolysis to <1 hour. No reduction or alkylation is required, nor is the critical filtration step required when using immobilized resins that are purported to speed up proteolysis.
Non-enzymatic chemical modifications such as deamidation, disulfide bond scrambling and oxidation negatively affect efficacy of biotherapeutic proteins. Peptide mapping is the primary analytical tool used to monitor these modifications. Unfortunately, the steps involved in peptide mapping sample preparation are also a source of post-translational modifications (PTMs).
To address these problems, we developed a sample preparation procedure according to which all sample preparation steps are performed at acidic conditions. Using this approach, we achieved robust digestion at acidic conditions while suppressing deamidation and disulfide bond scrambling.
Optimization of instrument performance
Data generated by scientific instruments and decisions based on that data depend on the instruments performing consistently. Users monitor parameters such as LC retention time, peak width and height while also monitoring MS parameters including accuracy, resolution, signal to noise, sensitivity (LOQ and LOD) and dynamic range. To this end, the 6 x 5 LC-MS/MS Peptide Reference Mixture was developed that will assess both LC and MS parameters.
Adequate monitoring of instrument performance for proteomics studies requires equally complex reference material. Whole cell protein extracts provide the necessary sample complexity. Preparation of such reference extracts is a challenging task because the extracts must meet a number of design requirements: compatibility with LC/MS, high protein integrity, and compatibility with common sample preparation methods such as proteolysis, PTM enrichment and mass-tag labeling. To meet these requirements the yeast and human whole cell protein extracts were developed.
These webinars are designed to support your experimental needs. If you still have questions about your bottom-up proteomics workflow, our Technical Services Team can help you troubleshoot your way to success.