Herein is described novel means of protein sequencing via sequential terminal chemical degradation (1, 2). The essence and novelty of the invention is that the cleaving reagent, and thus the resultant severed and modified amino acid residues, are affixed to a support(s) such as a biochip or beads.

To initiate the process the protein is put into Well 1 and the appropriate processes are effected. Subsequently the liberated protein, now shortened by one amino acid, is transferred to Well 2. This cycle of processing and transferring continues until, at the end thereof, each well contains an Edman reagent-amino acid derivation.

A simplification of this sequence would be to preposition the required alkali for the Edman reaction, perhaps in dry form, in each unreacted well. Also, while the final Edman reaction product is shown, it may not be necessary to convert the intermediate ATZ-amino acid derivative (see fig. 1 of ref. 1).

When utilizing the C-terminal sequencing process of Figure 1 in Reference (2), the sequencing reagent affixed to the support would be 2-(bromomethyl)naphthalene or equivalent alkylating agent. However, this reaction scheme may not be applicable do to the excess thiocyanate from the final simultaneous cleavage and activation step.

The next step is the identification of the amino acid derivatives in the wells, and preferably this is done with fluorescently labeled antibodies. One could reduce the number of distinct labels required if multiple identical parallel reaction sets were preformed, followed by probing each set with labeled antibodies directed to a distinct group of amino acids. Alternatively labeling could be done combinatorially has described in Fig. 5 & claims 9-12 of US Patent Application 20030036073 (4). A less preferred, yet potential means of detection is via mass spectrometry.

A significant potential advantage of this sequencing process is that loss of signal-to-noise ratio (SNR) due to incomplete primary reactions, might be avoided due to the ability to wash unreacted proteins from those reacted and bound to the support. Thus read lengths of 100 or more residues may be possible.

1) N-terminal Protein/Peptide Sequencing

2) Carboxyl-Terminal Sequencing Methods - Shively, John

3) Lab-On-A-Chip.com

4) Matrix Sequencing: A novel method of polynucleotide analysis utilizing probes containing universal nucleotides, Saba JA, US Patent Appl 20030036073, Filed Feb 2003

5) Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies. Yau, et al Biotechnol Adv. 2003 Oct;21(7):599-637 (also see MedLine link of "Related Articles")

6) Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features. Ruff-Jamison, et al J Biol Chem. 1991 Apr 5;266(10):6607-13

7) Molecular modeling and site-directed mutagenesis of an anti-phosphotyrosine antibody predicts the combining site and allows the detection of higher affinity interactions. Ruff-Jamison S et al Protein Eng. 1993 Aug;6(6):661-8

8) Requirement for both H and L chain V regions, VH and VK joining amino acids, and the unique H chain D region for the high affinity binding of an anti-phosphotyrosine antibody. Ruff-Jamison, et al J Immunol. 1993 Apr 15;150(8 Pt 1):3389-96

9) Generation of monoclonal antibodies against phosphotyrosine and their use for affinity purification of phosphotyrosine-containing proteins. Frackelton, et al Methods Enzymol. 1991;201:79-92. No abstract available.

James Saba:
Is reaction of lysine or arginine with the Edman reagent reversible? Is there ever a time, or is it even possible to selectively block lysine amines?

US Protein Sequencer:
The only side chain to which Edman reagent attaches hard enough not to come off in the acidic cleavage conditions is Lys. The problem of blocking Lys prior to sequencing is that most such reagents would also block the amino terminal group and thus defeat the Edman reaction. O-methyl isourea which converts Lys to homoarginine is an exception. We used it long ago to run Edman reaction on "native" soybean trypsin inhibitor. I have not heard of its use in sequencing of denatured proteins.

Additional Comments

There are many different recombinant antibody libraries available out there. By combination of a good library with the right selection strategy, you can almost isolate antibodies to all kinds of antigens. For example, there are antibodies specific to carbohydrates, which is very non-immunogenic.
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Your protein sequencing idea is certainly interesting! My best guess is as follows; It is highly likely that one can create antibodies against Edman derivatives with medium affinity. These molecules are however so small that high affinity antibodies are very difficult to make: on the other hand, derivatisation does help quite a bit. You could see in my paper that our monoclonal against 3-methylindole had only about 11% cross-reactivity against indole so one additional methyl group really did make a difference. However, even 11% cross reactivity may be too much in your application and create false signals. So specificity could be a problem concerning for example differentiation between Ala and Gly.
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I've only had a chance to glance at it, but that looks really cool....fast and easy as far as I can tell off the cuff. The biggest problems we've had with alternative methodologies have been in solvent choices/volubility. Your antibody detection method may be cost prohibitive, considering sequencing could require addition of all 20 antibodies to each/some wells, not to mention the antibodies would (I'm guessing) have to be 1) of pretty high quality to minimize cross-reactivity, and 2) in some way quantifiable so as to alleviate the problems caused by proline. You have a good idea, though.
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Thank you fort the interesting link to your new concept dealing with protein sequencing. I agree with you that such a method represents a fantastic progress facilitating fast sequencing of proteins applicable to a high throughput format. Basically it should be possible to develop specific binders to any protein and small molecule using phage surface display technology as stated in the review "High-throughput applications of phage display in proteomic analyses" which will appear in one of the next issues of TARGETS. However, such a task would requires a major effort based on high throughput screening of combinatorial libraries, selection of ligand panels and validation (which represent the most important step) and would also require a major financial effort. Theoretically there are no reasons to assume that such an approach would not work.
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I have discussed the use of antibodies with some of my colleagues within the past two weeks. It seems that some people have attempted to perform Edman's degradation on chip before, but not with the use of antibodies.
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We have 5 different colors of Quantum Dots (emission wavelengths) for our Streptavidin Conjugates. They are 525, 565, 585, 605 and 655 nm. When using the optimal filter sets for the quantum dots (available from Omega Optical and Chroma Technology), they are 2 - 10 orders of magnitude brighter than organic dyes.
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When we're detecting fluorescently-labeled proteins from human serum using antibody microarrays, we can get down to about 1 ng/ml. We now have amplification methods working well that get us down to 100 pg/ml or so. It also depends on the quality of the antibody.
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We use concentrations of around 500 ug/ml for the capture antibody. Spot size in principle doesn't matter as long as your scanner resolution samples it sufficiently. (i.e. for a 100 um diameter spot, need a resolution of 10 um or so.)
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To answer your question about generating antibodies to all 20 different amino acid residues, I think some will be easier than others. For example, Trp, and Tyr would be the easiest among all, while Gly and Ala would be the toughest. However, there would be ways to circumvent the problems. All in all, I think it is feasible but highly depends on time and resources.