Samples to Spectra to Protein IDs
How we get from biological samples to protein identifications?
Every sample that we receive must be prepared for mass spectrometry, analyzed with appropriate mass spectrometric methods, and the resulting data analyzed appropriately. There are many routes to this process that depend on the goals of a particular experiment. Here, we outline some of the steps that are generally common to most sample workflows.
Clean-up and Preprocessing
Samples derive from many sources, including blood, tissue, cell culture, and other material (we have even performed experiments on lizard eggs). We commonly receive samples in matrices that are incompatible with down-stream processing steps. For this reason, we must often take several steps to "clean-up" the sample prior to further steps. These steps may include gel electrophoresis, buffer exchange by dialysis, concentration of the sample by spin-filter or lyophilization, etc. The specific steps performed depend on the overall design and goals of the proteomics experiment.
The goal of many proteomics experiments is to examine the structure and function of a protein of interest. Proteins, in their intact form, have very complex, folded structures that make any sort of analysis by mass spectrometry very difficult (fig. 1). For this reason, proteins and mixtures of proteins are typically unfolded and broken down into smaller, more manageable pieces prior to any analysis by mass spectrometry. Digestion protocols for proteins existing either in-solution or in-gel are very common and consist of the following steps:
- Reduction and Alkylation
Denaturation is the process of unfolding a protein from its intact, quaternary structure to its single-stranded polypeptide form. The addition of a chaotropic agent, such as guanidine hydrochloride or urea, in combination with heat is the most common method of action. SDS-PAGE gels also inherently denature proteins, thus serving as a common and convenient step to both clean-up and denature proteins in one step.
Reduction and Alkylation
Even after being denatured, a protein may retain some structure due to disulfide bonds. The breaking of these covalent bonds occurs during a reduction step and the capping of the resulting free sulfur occurs during an alkylation step (fig. 2). Alkylation is performed so that disulfide bonds cannot reform, leaving the protein in a completely denatured state. Common reduction and alkylation agents include dithiothreitol and iodoacetamide respectively.
The process of digestion utilizes proteolytic enzymes that cleave peptide bonds at very specific amino acids along the polypeptide chain (fig. 3). This yields a series of smaller peptides that are now ready for analysis by mass spectrometry (fig. 4). Protein digestion prior to mass spectrometry analysis is a very common proteomics tool and can be utilized for a variety of applications including sequence determination, post-translational modification analysis and biomarker discovery/verification.