Reasonable Expectations

N-Blocked Proteins Reasonable Expectations:

About 50 % of all proteins submitted for sequence analysis do not provide an N-terminal signal due to natural (usually natural acetylation) or artifactual blocking due to exposure to chemical reagents during purification or other processing. These chemical modifiers include heavy metal ions, aldehydes, cyanates from Urea etc. The main reason for our requirement of a 10 cycle minimum is to evaluate roughly the amount of protein present (contributing to the background) In the case of N-blocked protein. It is also to determine if there is a faint signal present (in the case of an artfactually N-blocked protein). Ten cycles is usually the minimal number of sequencing signal per cycle data to allow a decision of whether there is sufficient protein available for attempted internal sequencing using our unique methodology.

We use a two step method which will provide internal sequence data on most samples. First , we will do an in-situ CNBr digestion on the immobilized protein PVDF blot. (Note: All samples must be immobilized on 0.2 micron pore size PVDF for internal sequencing!) The sequence analysis of this digestion will provide information from a mixture of peptides. Very often, especially on smaller proteins below 50K where there are few Methionine residues, a dominant sequence will arise due to an easily cleaved or more exposed Methionine. This dominant sequence that can be largely deduced owing to it's stronger signal. This sequence frequently can be read well enough to help identify the protein from database search methods.

If this initial work does not provide a dominant signal, then a second method is employed using the data provided from the CNBr digestion. The initial CNBr digest sequence data is scrutinized to determine the positions and yields of Proline residues. Proline is the only amino acid that does not become covalently blocked by the reagent O-phtalaldehyde (OPA), thus providing a chemical selection of the Proline containing peptide from the mixture. No effort is made to isolate any of the digestion peptides by HPLC or other techniques that typically loose 70 to 90% of the peptide. Rather, the application of OPA at a known Proline position in the digest will block all peptides except the peptide containing Proline thus, chemically selecting the peptide, not physically removing it from the digest (Brauer, A.W., Oman, C., and Margolis, M.N. Analytical Biochemistry 137; 134-142, 1984.) This method usually increases by about a factor of ten the sensitivity in isolating an internal sequence and can usually be successful on a few picomoles (5 to 10 picomoles) of protein. The criterion for success usually calls for 1.0 picomole of Proline producing a signal at least 50% over background as seen on the initial CNBr digestion. We encourage you to provide your target in duplicate bands of approximate equal amount. This will ensure that it will be possible to perform both procedures for the best possible chance of obtaining a clean internal signal. Although it is possible to do a CNBr digest on a single submitted sample after it has been through a sequence run (and found to be N-terminally blocked), the best possible situation for obtaining a good internal sequence is achieved from freshly supplied duplicate bands.

If you have any questions about this methodology please: E-mail: Proseq@tiac.net

or call (978) 213-9911

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	N-Blocked Proteins Reasonable Expectations: About 50 % of all proteins submitted for sequence analysis do not provide an N-terminal signal due to natural (usually natural acetylation) or artifactual blocking due to exposure to chemical reagents during purification or other processing. These chemical modifiers include heavy metal ions, aldehydes, cyanates from Urea etc. The main reason for our requirement of a 10 cycle minimum is to evaluate roughly the amount of protein present (contributing to the background) In the case of N-blocked protein. It is also to determine if there is a faint signal present (in the case of an artfactually N-blocked protein). Ten cycles is usually the minimal number of sequencing signal per cycle data to allow a decision of whether there is sufficient protein available for attempted internal sequencing using our unique methodology. We use a two step method which will provide internal sequence data on most samples. First , we will do an in-situ CNBr digestion on the immobilized protein PVDF blot. (Note: All samples must be immobilized on 0.2 micron pore size PVDF for internal sequencing!) The sequence analysis of this digestion will provide information from a mixture of peptides. Very often, especially on smaller proteins below 50K where there are few Methionine residues, a dominant sequence will arise due to an easily cleaved or more exposed Methionine. This dominant sequence that can be largely deduced owing to it's stronger signal. This sequence frequently can be read well enough to help identify the protein from database search methods. If this initial work does not provide a dominant signal, then a second method is employed using the data provided from the CNBr digestion. The initial CNBr digest sequence data is scrutinized to determine the positions and yields of Proline residues. Proline is the only amino acid that does not become covalently blocked by the reagent O-phtalaldehyde (OPA), thus providing a chemical selection of the Proline containing peptide from the mixture. No effort is made to isolate any of the digestion peptides by HPLC or other techniques that typically loose 70 to 90% of the peptide. Rather, the application of OPA at a known Proline position in the digest will block all peptides except the peptide containing Proline thus, chemically selecting the peptide, not physically removing it from the digest (Brauer, A.W., Oman, C., and Margolis, M.N. Analytical Biochemistry 137; 134-142, 1984.) This method usually increases by about a factor of ten the sensitivity in isolating an internal sequence and can usually be successful on a few picomoles (5 to 10 picomoles) of protein. The criterion for success usually calls for 1.0 picomole of Proline producing a signal at least 50% over background as seen on the initial CNBr digestion. We encourage you to provide your target in duplicate bands of approximate equal amount. This will ensure that it will be possible to perform both procedures for the best possible chance of obtaining a clean internal signal. Although it is possible to do a CNBr digest on a single submitted sample after it has been through a sequence run (and found to be N-terminally blocked), the best possible situation for obtaining a good internal sequence is achieved from freshly supplied duplicate bands. If you have any questions about this methodology please: E-mail: Proseq@tiac.net or call (978) 213-9911