Determination of Primary Structure

The procedure for determination of the primary structure of a protein is outlined in the figure below. The first step (1. in the figure) is to determine the molecular weight of the intact protein. This can be done using either FAB, electrospray, or matrix assisted laser desorption mass spectrometry. Knowledge of the molecular weight of the intact protein is necessary to be sure that the entire protein is accounted for in analyzing fragments of the protein. The next step is to cleave the protein into smaller fragments and determine the amino acid sequence of each fragment (2. in the figure). If the protein contains cysteine residues, it is normally subjected to reduction and carboxymethylation of the cysteine sulfhydryl groups prior to cleavage (alternatively carboxamidomethyl or pyridylethyl derivatives may be prepared). Cleavage is then performed either chemically or enzymatically.

Large proteins are usually cleaved in two stages: a first cleavage is performed at relatively low abundance sites (step 2a, e.g. cyanogen bromide cleavage at methionine residues) to yield large fragments which are separated and then cleaved at more abundant sites (step 2b, e.g. trypsin cleavage at lysines and arginines). Smaller proteins can be initially cleaved at higher abundance sites. The cleavage mixtures are then separated into simpler mixtures by HPLC (step 2c). Unlike with Edman degradation where it is necessary to individually purify each degradation product, the mass spectrometric approach can be carried out with mixtures of cleavage fragments. The fractionation into simpler mixtures is done to overcome potential competitive effects in the ionization process to increase the probability of observing all of the fragments in the mass spectrometer. The fragment mixtures are first subjected to single stage mass spectrometry to determine the molecular weights of the fragments (step 2d) and then analyzed by tandem mass spectrometry (step 2e) to determine the amino acid sequence of each fragment.

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Biomolecular Mass Spectrometry Facility Department of Pharmacology Medical University of South Carolina 173 Ashley Avenue, CRI 305 Charleston, SC 29425	Telephone Numbers: 843-792-5849 (CRI 305) 843-792-2471 (department office) FAX: 843-792-2475