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Protein Identification

Here we are referring to relative pure proteins, such as recombinant proteins for CryoEm or X-ray crystallography studies. Sample in solution (preferred) or gel bands from 1D PAGE separation are digested with a specific protease, typically using trypsin, and the resulting peptides are analyzed by mass spectrometry (MS). In our lab, we can analyze using Matrix Assisted Laser Desorption Ionization (MALDI) MS or separate the tryptic peptides using nano-flow liquid chromatography and detected via electrospray ionization (ESI)-MS analysis. In either case, peptide species are distinguished by measuring the ionized mass-to-charge (m/z) values and fragmenting them using tandem MS (also stated, “MS/MS”), to obtain amino acid sequence information of individual peptides belonging to specific proteins. Taken together, the intact peptide and fragment data are then used to computationally search databases specific to the organism of interest and thus identify proteins in the original mixture. The goal being to get enough sequence information to identify the protein, and not necessarily get complete sequence coverage like in peptide mapping experiments. For recombinant proteins, we ask for amino acid FASTA files, including any His-tags or similar sequence modifications. It is also important to let us know how the proteins were produced so we can look for everything in the sample. For example, if you are expressing a Human protein in E. Coli, we need to know to look for potential contamination by bacterial proteins.

Figure 1. A typical workflow for protein identification. A) A protein isolate is separted via PAGE gel electrophoresis and then extracted to obtain a relatively pure protein. The protein is then digested using trypsin to produce peptides. These peptides are analyzed via LCMS and provide a MS spectrum that is characteristic of the isolated protein. B) To identify the protein from the acquired MS spectrum, a library of proteins is searched with hypothetical trypsin digested peptides that give a predicted MS spectrum. A match between the predicted MS spectrum to the acquired mass spectrum indicates the identified protein.