After each cycle of Edman chemistry, the PTH-amino acid is analyzed by the on-line amino acid analyzer. Each cycle of sequence is presented as a chromatogram of the PTH amino acid. Sequences were called by comparing amounts of amino acids present in each cycle. In cases where more than one amino acid were seen in a cycle, the amino acid whose amount most closely corresponded with the rest of the sequence (based on amount and operator judgement) was called. Residues that have reduced yields (serine and threonine) were taken into account.
Cycles where no amino acid was seen are denoted with “X”. It should be noted that if your sample was not reduced and alkylated, a cycle designated by X might actually contain a cysteine residue. Alternatively, some other residue that does not appear on the PTH-amino acid standard may be present (phosphoamino acid, glycosylated amino acid, lipid-modified). Cycles where the sequence ended (the end of the peptide/protein were reached) are so denoted.
Understanding the definitions of certain terms may be useful to you in evaluating and understanding your data. These terms are important in allowing you to know the efficiency of the sequencing experiment.
Initial yield (IY): This is the amount of material estimated to be present in your sample. IY is reported in amount of amino acid detected in cycle one or in percent, where the amount of material analyzed was known prior to the experiment. For example, if we load 100 pmol of a protein onto the bi-phasic column and we detect 50 pmol of the first amino acid we calculate an Initial Yield of 50%. If the amount of material analyzed is not known we cannot report a percentage. Initial yield is never 100%. The value for IY is determined by the nature of the Edman Chemistry and efficiency of the protein sequencer.
Repetitive yield (RY): Repetitive yield is a measure of the efficiency of the Edman Chemistry during the sequencing run. The value is reported in %. Acknowledging that the Edman chemistry is never 100% efficient, the RY is also affected by condition of the protein sequencer (how well is it tuned and maintained), the nature of the primary structure of the protein or peptide being sequenced and the extraction-efficiency of the individual PTH amino acids from the sequencing column to the conversion flask. Generally, the IY and the RY for a liquid sample are higher than the same sample on PVDF membranes. This is probably due to the reduce access of sequencing reagents to the membrane-bound sample and the greater difficulty in extracting derivatized amino acids from the hydrophobic membrane. The IY and the RY has a direct effect on the maximum number of cycles of sequence possible.
Carry-over (lag): Lag is the detection of the amino acid from the previous cycle of sequencing in your current cycle. Lag is variable and is affected by the condition of the protein sequencer and the primary structure of the sample and the matrix in which it is presented to the Edman chemistry (liquid on C18 vs PVDF). Lag tends to build-up over longer runs and can eventually interfere with accurate amino acid assignments. It is related to RY in that the higher the RY, often the lower the lag.