| N-terminal Sequence Analysis |
| N-terminal sequence analysis of a protein or peptide consists of repetitive cycles of the Edman chemistry followed by PTH analysis using microbore HPLC. One cycle of the Edman chemistry and the resulting PTH chromatogram represents identification of one amino acid. Each cycle of the chemistry takes 30-50 minutes depending on the instrument used. If the protein has a blocked N-terminus, no data can be obtained. It has been estimated that 40 - 70% of all naturally occurring proteins are N-terminally blocked. Chemistry: Automated Edman degradation chemistry consists of three steps, a) coupling of phenylisothiocyanate (PITC) with the alpha-amino group of the protein/peptide at pH 9-10 to form a phenylthiocarbamyl (PTC) group, b) cleavage by anhydrous acid (trifluoroacetic acid (TFA) to generate an anilinothiazolinone (ATZ) amino acid and c) conversion of the ATZ derivative to the more stable phenylthiohydantoin (PTH) derivative. Solvent extractions between the above steps wash out by-products and excess reagents. Finally, the PTH amino acids are analyzed by microbore HPLC. It should be noted that cysteines are destroyed by the chemistry and therefore cannot be identified unless reduction and alkylation is performed. We routinely monitor for modified the cysteine derivatives carboxyamidomethylcysteine (CAMC), which is generated during sample preparation and propionamide cysteine (PAMC), which is formed through a side reaction of free acrylamide with cysteine during electrophoresis. Post-translational modifications can present their own individual problems. Instrumentation: The Service has two automated protein sequencers. One Applied Bioystems (ABI) 494 gas-phase/pulsed-liquid Procise-HT sequencer is equipped with a 190C PTH analyzer, and the other ABI 494 pulsed-liquid Procise-HS sequencer is equipped with a capillary 140B PTH analyzer. The ABI 494HT is used for moderate levels (1-10 pmol) of HPLC purified peptides as well as low-level PVDF-bound proteins. The ABI 494 HS is used only for very low-level HPLC purified peptides (0.1-1 pmol). All instruments analyze peptides suspended on a glass fiber disc using a polymer (polybrene) to retain samples through hydrophobic interactions. Yields and expectations: The performance of each instrument and individual sample is characterized by initial and repetitive yields as well as carryover. The initial yield (typically 50-80% based on standard proteins) refers to the quantity of amino acid recovered in the first cycle of the Edman chemistry and is expressed as a percentage of the total sample analyzed. The repetitive yield, typically 90-99%, represents the recovery of the PTH amino acid after each cycle of the chemistry, and is dependent on the instrumentation as well as the individual characteristics of the sample. Carryover (or lag) is the amount of the previous amino acid present in the subsequent cycle. The expected number of amino acids which can be determined for a particular protein or peptide sample is dependent on its quantity, purity, and size. For example, 10 pmol of a 100 kD protein (on PVDF or solution) with an initial yield of 50% would yield 15-40 residues of usable sequence data depending on its structure and purity, while 10 pmol of an HPLC purified peptide fragment 20 amino acids long with an initial yield of 50% could probably be completely sequenced. |