Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6818—Sequencing of polypeptides
  • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

• This invention relates to methods and radiolabeled reagents for super-sensitive protein microsequencing and amino acid analysis. More specifically, methods of radiolabeling of Edman- type reagents and their use with multiphoton detection are disclosed.
• Biomedical applications require ever increasing sensitivity for the characterization of single organisms (virus, bacterium) or single cells. Especially important is the detection of differences in protein composition of normal vs. diseased cells, e.g. cancer cells.
• Multiphoton Detection enhanced 2D gel separation permits specific selection of a protein from a complicated mixture.
• the prior art limit of detection (LOD) for proteins is typically on the order of pg/ml or 0.1 fmole/ml.
• Recently, BioTraces, Inc.'s MPDTM Multi Photon Detection technique achieved 0.01 pg/ml or 0.5 attomole/ml sensitivity. Not only the detection, but also the quantitation of the protein is important, for example in the case of cancer markers.
• each target should be characterized, e.g. in the case of metastatic cancer cells which have to be distinguished from millions of virtually identical healthy cells.
• the ability to microsequence polypeptides and proteins is of increasing importance.
• the prior-art methods of microsequencing require a rather large amount of proteins, typically a few picomole.
• only attomoles of a given protein may be available.
• the proteins from hundreds and thousands of cells have to be pooled. This, however, means that in the prior-art only averages and not distributions of biologically important proteins can be measured and characterized.
• microsequencing is a method which also provides considerable redundancy and facilitates NSBB suppression. Methods are required which permit a major improvement (by a factor of a hundred and more) of sensitivity of polypeptides and proteins microsequencing.
• a supersensitive method for quantitation of radiolabeled, especially radioiodinated polypeptides and proteins uses MPDTM MultiPhoton Detection (MPD). MPD technology enhances high performance liquid chromatography (HPLC) and permits reliable quantitation of some radiolabeled analytes down to 0.1 attomole/sample.
• MPDTM MultiPhoton Detection MPDTM MultiPhoton Detection
• proteins are not self-replicating template molecules like DNA and there is no way to amplify a protein in vitro other than to isolate and amplify the RNA transcript or gene that encodes it and then translate it in vitro. And even if this tedious procedure is to be used, which is only possible if the gene sequence is known, the copies may not be identical with the original due to post-translational modifications (e.g. chaperon-aided folding, glycosylation etc) that occur in vivo, but are difficult to detect and/or to reproduce in vitro or in different organisms. Therefor, the fundamental issue in protein analysis is the sensitivity of the methods to detect, analyze and manipulate the proteins.
• the invention provides methods of peptide and protein microsequencing using radiolabeled reagents to perform peptide and protein microsequencing at levels lower than 10 femtomole, preferably at a level lower than 1 femtomole.
• Bioreagents according to the invention are Edman-type reagents which have an aromatic chemical structure, a reporter moiety(moieties) that is or may be derivatized to include a radiolabel, a purity preferably better than 99.99% level, and an ability to react with amino acids by means of an active group such as isothiocyanate.
• the radiolabels are compatible with MultiPhoton Detection techniques. They may be selected from the family of EC emitters and positron/gamma emitters. They may be halides, including radioisotopes of iodine, including 123 I, 124 I, 125 I, and 126 I. They may be EC emitters from the lanthanides family or the platinides family.
• the isothiocyanate group is alternatively a substituent in an aromatic ring other than phenyl such as naphtyl, anthracenyl, pyrenyl, pyridyl and the like.
• the hydrogen atoms in the phenyl or other aromatic ring are preferably substituted with groups such as methoxy, hydroxy, and the like in order to facilitate radioiodination without significantly diminishing reactivity of the isothiocyanate moiety.
• the radioiodinated Edman reagent can be radioiodinated before or after the degradation cycle and before or after the separation step.
• the radioiodinated Edman reagent used in the degradation has a protected active group that is deprotected and labeled before the separation step.
• the active group is preferably a primary or secondary amino group.
• the linker is preferably an alkyl chain containing 1 to 30 carbon atoms terminated with a reporter group.
• the reporter group is preferably a primary or secondary amino group or an active group such as aldehyde, hydroxyl, carboxyl, sulfhydryl, phosphate, phosphorothioate, azido, and the like or combination thereof.
• the linker chain contains for example 1 to 15 heteroatoms, such as oxygen atoms.
• Radiolabeling may be achieved using [ 125 I]iodinated N-succinimidyl-3-(4-hydroxyphenyl) propionate (Bolton-Hunter reagent), [ 125 I]iodinated N-succinimidyl-3-(4-methoxyphenyl) propionate (modified Bolton-Hunter reagent), metal [ 125 I]iodide salt or salts for direct iodination in oxidizing conditions, [ 125 I]iodinated reagents and any type of chemistry used generally for chemical conjugation.
• the excess of the labeling reagent may be removed by simple physicochemical operations such as extraction, gel filtration, gel chromatography, precipitation, adsorption and the like.
• the product of the cleavage step may be reacted with the primary amine group of a supplemental reagent.
• the supplemental reagent may be radiolabeled, while the Edman reagent is unmodified.
• the method may be used for amino acid analysis comprising the following steps:
• HPLC high performance liquid chromatography
• TLC thin layer chromatography
• CE capillary electrophoresis
• GE gel electrophoresis
• a device comprises a spatially resolving MPD (SR-MPD) detector.
• the SR-MPD is for example initially placed around the HPLC column, typically in the middle ofthe column but is movable and it's movement is computer steered to follow up movement of the peak of analyzed Edman degradation product within a column.
• the SR- MPD may comprise a series of linearly arranged scintillator crystals each with the dedicated photodiode or avalanche photodiode readout.
• the SR-MPD movement is for example initiated after the detector registers at least 10 counts providing that the count rate is compatible with previously measured activity of Edman degradation product.
• SR-MPD movement may be initiated after the detector registers at least 10 counts providing that the count rate is compatible with previously measured activity of Edman degradation product.
• the SR-MPD movement is preferably computer controlled, so that the maximum count rate is at the center of the said SR-MPD.
• SR-MPD signal registration is for example performed several times with increasing resolution, preferably three times.
• the TLC may be TLC on silica gel, TLC on a Reversed Phase carrier.
• the TLC plate is analyzed by means of MPD instrumentation, preferably an SR- MPD device.
• the separation process is preferably performed simultaneously for many sequencing reactions in parallel.
• the number of sequencing reactions performed in parallel may be in the range 1-200.
• the migration of the Edman degradation product can be stopped when still inside the HPLC capillary, and an MPD Imager used to quantitate the distribution of radiolabeled Edman degradation product inside the capillary.
• An SR-MPD can be initially placed around the capillary, typically in the middle ofthe column but is movable and with movement computer steered to follow movement of the peak of analyzed Edman degradation product within the capillary.
• the SR-MPD may be linear and comprise a series of linearly arranged scintillator crystals each with the dedicated photodiode or avalanche photodiode readout.
• a linear SR- MPD may have movement initiated after the detector registers at least 10 counts providing that the count rate is at compatible with previously measured activity of Edman degradation product.
• the linear SR-MPD movement is preferably computer steered so that the maximum count rate is at the center ofthe said linear SR-MPD.
• Fig.1 Edman degradation cycle (prior art)
• Fig.2 Linearity of HPLC/MPD measurements of a mixture of steroidal hormones; the results for dilutions down to a factor of 1:1024 are shown. The lowest point corresponds to 0.02 attomole/sample.
• Fig.3 Reproducibility of HPLC/MPD measurements of a few attomoles/sample of a mixture of estradiol and testosterone; the total count at the peaks is plotted.
• Fig.4 Well resolved peak of insulin for concentration of 5 attomole/sample.
• Fig.5 Linearity of HPLC/MPD for insulin.
• Fig.6 Reproducibility of HPLC/MPD for insulin.
• Fig.7 Radioiodination of Edman reagent
• Fig.8 Radioiodination of Edman reagent
• Polypeptide or protein as used here means proteins, short peptides, and other compounds comprising amino acids linked by peptide bonds. These compounds may be modified, combined, complexed, derivatized, and so on.
• Edman reagent or Edman type reagent means any reagent that is able to couple to all types of amino acids, and to sequentially bind to and cleave the amino acids from a polypeptide to form an Edman derivative, the derivative of each amino acid being able to be resolved on separation as a distinctive peak.
• the reagent must be able to do this with all amino acids at high efficiency (e.g. greater than 90%, preferably greater than 99%), and with essentially no side products that would interfere with resolution and detection ofthe peak.
• Phenyl isothiocyanate (PITC) degradation (Edman, 1950) is the most efficient method for the sequencing of both small peptides and large proteins. The basics of this method are illustrated in Figure 1. There have been many modifications to the basic method, such as the liquid phase, or spinning cup sequencer (Edman and Begg, 1967), and an automated solid phase sequencer has also been described (Laursen, 1971). Both of these machines answered some of the limitations of the manual method. A gas-phase sequencer (Hunkapiller and Hood, 1978) is the current method of choice for sequencing both peptides and proteins. However, a rapid manual method is still used in some laboratories for chemical characterization of peptides and proteins isolated and purified chromatographically.
• the MPDTM system can quantify amounts of compounds as low as a hundred molecules, i.e. at sub-zeptomole ( ⁇ 10 ⁇ 21 mole/sample) levels. See U.S. 5,866,907 and U.S. 5,854,044, incorporated herein by reference.
• HPLC/MPD MPD readout
• these inventions provide a sensitive analytical technique which is applicable to separation, detection and quantitation of most analytes. For important biomolecules (neurotransmitters, steroids, insulin) limits of detection are about 1000-fold better than prior-art techniques. Sensitivity of 0.1 attomole/sample (10 "19 mole) sensitivity has been demonstrated.
• LODs Limits of detection
• HPLC/MPD Chromatographic studies depend on the measurement of analytes which are often present at very low levels and current detectors are not sensitive enough for many applications. Even such sensitive methods as electrochemical detection and mass spectroscopy are often not sufficiently sensitive.
• the most sensitive chromatographic detection method compatible with Edman reagent at the level of a few hundreds of attomoles is laser-induced fluorescence (LIF).
• BioTraces, Inc. developed an ultrasensitive method for I labeled testosterone and estradiol.
• the separation of iodinated steroid hormones (Amersham) was performed using a Perkin Elmer instrument with Binary LC Pump 250, PE Pre Column Scavenger and C 18 Cartridge Column.
• the individual compounds were dissolved in a methanol/water (9:1) mixture and dilutions from 100 pg/ml to about 100 fg/ml were prepared. Samples were 10 microliters, i.e. 1 fg/sample for the highest dilution.
• HPLC/MPD excels in analysis of peptides and proteins. Attomole sensitivity has been demonstrated for a radio-iodinated peptide-insulin (6,000 Daltons).
• a solution of iodinated porcine insulin (7 x 10 "16 mole) was prepared by diluting a commercial 125 I labeled insulin (DuPont) in water and acetonitrile (50:50).
• Replicants were prepared by diluting commercial I labeled insulin in 50:50 wate ⁇ acetonitrile or urine (human female): acetonitrile in buffer and urine before distribution as 100 1 aliquots. Serial dilutions at a factor 1/4, starting at 1.75 x 10 "16 mole down to sub-attomole were used.
• Reverse phase HPLC analyses were preformed on 100 1 samples, injected manually, at ambient temperature on an Alltech Kromasil C8 column (25 x 0.46 cm, particle size 5 m ) with a guard column of the same packing.
• Eluent A was 0.1% trifluoroacetic acid (TFA) in water and eluent B was 0.1% TFA in acetonitrile.
• the solvent gradient used was 20% B increasing linearly at a rate of 2.5% a minute to a final concentration of 60% (held isocratic for 10 minutes).
• the flow rate was 1.0 ml/min and fractions were collected every minute for 20 minutes for counting by MPD.
• Figure 4 demonstrates a very well separated insulin peak for attomole/sample concentration of insulin, with the background below attomole/sample.
• the linearity of HPLC/MPD is shown in Figure 5.
• the reproducibility for dilute sample (a few attomoles/sample) is shown in Figure 6.
• the limits of detection when using HPLC/MPD for appropriately radioiodinated compounds are more than 1,000 -fold better than prior-art chromatographic methods.
• TLC thin layer chromatography
• an increase in sensitivity for polypeptide sequencing is achieved by:
• the invention permits peptide/protein sequencing at better than 100 attomole level, i.e. more than 1000-fold improvement over prior art methods, and to sub-attomole level.
• PITC PITC-amino-acid derivatives are relatively stable, with up to 60- min incubation in an aqueous buffer at room temperature.
• Another reagent is OP A, which produces a highly fluorescent adduct but OPA-2-mercaptoethanol isoindole derivatives of lysine degrade up to 30- 40%, histidine up to 20%, and many others up to 10% for the same time period.
• thiols e.g., ethanethiol or 3-mercapto-l-propanol, degradation rates are partially reduced.
• a second disadvantage is the failure of OPA to react with secondary amines, proline and hydroxyproline.
• Na hypochlorite is used as an oxidant.
• the detection of these amino acids following this oxidation step has found only limited application in post-column detection systems and the quenching characteristic of Na hypochlorite and its oxidation of other amino acids limits sensitivity and makes the OPA system less desirable.
• losses with OPA are compared over a 12-h period, decreases range between 2 and 50%. In those cases where apparent increases were noted, i.e., glycine and proline, co-elution of breakdown products with the derivatized amino acids was found responsible for these increases.
• FITC may decompose during the degradation.
• the reagent carries many functional side groups that can give rise to many side reactions. Consequently, many extra spots are visible on the thin-layer sheets which obscure the degradation result. Therefore, higher quantities of the peptide must be subjected to the sequence analysis than with DABITC or PITC.
• the second problem can be largely diminished when using more sensitive detection means.
• Each purification method leads to a certain, often large, loss of studied analyte.
• more specific methods of purifications can be used, because one is not limited by the need to extract almost all present material.
• the problem of differential mobility is drastic when using pre-column derivatization, e.g. when using fluorophores as labels.
• the molecule modification by an attachment of a single atom of radioiodine is much lower, and the differential mobility either negligible or can be calibrated.
• the "two color" labeling available with 125 I and 123 I can be used to diminish the uncertainties due to nonspecific losses.
• isolation of the protein is achieved by blotting appropriate gel on the membrane.
• Modern gas-phase sequenators allow direct use of the blot for sequencing.
• a common problem is insufficient amount of the protein transferred to the membrane.
• multiple blotting or repeated electrophoresis might be necessary. This leads to multiple layers of blotting membrane in the chamber, and as a result less reliable reaction yields.
• Use of much smaller amounts of the proteins or mixture thereof significantly improves resolution ofthe gel, and thus purity ofthe final blotted sample.
• the yield of each step is such that the amount of cleaved amino acid decreases in an exponential way as the sequencing proceeds. Therefore, the Edman degradation has to be stopped after a certain number of cycles, depending on the amount of starting material. Sometimes, the number of cycles performed is not enough to allow unambiguous and/or rapid identification ofthe protein (e.g. impossibility to identify variants ofthe protein such as splice variants, post-transcriptionally modified variants, or design of too small or too degenerate oligonucleotides to screen a cDNA library). In some instances, the complete sequence could even be achieved, and the need of still difficult C-terminus sequencing would become dispensable.
• proteins present in smaller samples could still be reliably identified. For instance, identification of proteins present in precious samples like tissue biopsies or biological fluids could be carried out from smaller samples.
• smaller sample could mean less starting material and less reagents to prepare this material.
• the invention permits study of proteins poorly expressed in cells or tissues like transcription factors or variants of proteins that are poorly represented in a tissue (e.g. mutated oncogene in a tumor sample, in which the majority of the sample protein is unmutated).
• the level of global protein expression termed "proteome”
• proteome can be assayed by means of two-dimensional gel electrophoresis.
• 10 micrograms of protein can be loaded on such gels, which can be resolved into as many as 5000 spots of higher intensity.
• many important proteins are present in low or undetectable levels by conventional methods.
• the MPD-enhanced detection of this set of poorly expressed proteins has now become possible, as well as their purification from 2D-gels.
• MPD-enhanced Edman degradation then allows the identification and sequencing of these proteins, which has remained an unreachable goal for the man ofthe art using existing methodologies.
• the primary structure of proteins or peptides can be elucidated in two ways: a direct method consists in sequencing the protein using sequencing methods, the most widely used being the Edman degradation. The second, and indirect method, consists in determining the sequence ofthe gene or its transcript encoding this protein, with the assumption that this gene has been identified and that its sequence has been previously elucidated.
• a conceptual translation based on the knowledge ofthe genetic code provides the sequence ofthe protein. However, the actual sequence of the protein may differ from the sequence derived by conceptual translation for a variety of reasons: first the genetic code is not universal; some organisms or organelles (e.g.
• mitrochondrium use variants of the code used by eukaryotes; the genetic code may even be altered in a given cell under some circumstances (e.g. stop codon suppressor phenotype in bacteriophage-infected bacteria).
• the exact sequence of the transcripts of a given gene is not fully predictable: in eukaryotes, they may be differentially spliced according to tissues or time of expression, or even edited in some occasions, so that the mere knowledge of the gene sequence is often insufficient to deduce the protein sequence.
• proteins can undergo, after translation, some of many possible post-translational modifications of their primary structure that may be either definitive or time-dependant (e.g. protein cleavage, myristilation, deamidation, glycosylation, phosphorylation).
• Pre-column derivatization is an important method in peptide/protein sequencing.
• derivatizing agents used in chromatography such as luminophores and fluorophores, we call these agents radiophores.
• the first process takes advantage of the presence of an activated aromatic ring in some natural amino acids (tyrosine and histidine) and relies on electrophilic aromatic substitution with iodide ion in the presence of an appropriate oxidizing agent such as chloramine T.
• This method permits the radioiodination of two out of 21 amino acids. It is, very important however, because it permits one to calibrate the losses that occur during the process, e.g. tyrosines and histidine can be radioiodinated with 123 I, and total activity measured before Edman degradation process. Measurement, after each step of process, permits reliable estimate ofthe step yield.
• the main novelty of the disclosed method is to radiolabel, preferably radioiodinate the Edman reagent which during the microsequencing of peptides/proteins is reacted with N- terminal amino acid.
• radiolabel preferably radioiodinate the Edman reagent which during the microsequencing of peptides/proteins is reacted with N- terminal amino acid.
• Electrophilic aromatic substitution is a very effective method. Unfortunately, it requires that the iodide ion is oxidized to iodine atom. Two atoms form a molecule of iodine which is the immediate source of the electrophile. Molecular iodine is highly volatile, and thus in the case of radio-isotope, requires special safety precautions.
• the aromatic ring of the iodinated compound preferably contains an electron donating group such as hydroxy, alkoxy or amino to facilitate an electrophilic reaction.
• Pre-column Labeling In a preferred method, where the least possible modification of the existing procedure is used, 4-[ 125 I]-iodophenyl isothiocyanate and I25 3-[I]-iodophenyl isothiocyanate can be synthesized and substituted for PITC. 4-[ I]-iodophenyl isothiocyanate may be obtained by isotope equilibration of 4-iodoaniline, and subsequent conversion of the amino group into isothiocyanate with carbon disulfide in the presence of dicyclohexyl carbodiimide (DCC)(Burrel et.al. 1975).
• DCC dicyclohexyl carbodiimide
• the reagent was therefore inappropriate for microsequencing as evidenced by its disuse for over two decades. Higher purity and yields are accomplished according to the invention.
• 3-[ I] -iodophenyl isothiocyanate may be obtained in one step by electrophilic substitution of the tri- «-butylstannyl group in meta position of the PITC with I in the presence of oxidative reagent (Iodo-Gen)(Ram et. al. 1994). 3-[ 125 I]-iodophenyl isothiocyanate in this report was used for antibody labeling without any reference to protein sequencing.
• any combination of heteroatoms can be present in the aromatic ring and the examples of such systems include: N-alkyl pyrane, thiophene, pyrimidine, pyridine, indol, phenantrolines, and the like, where arrangement of the iodo and isothiocyanyl substituents, possibly together with other substituents, would result from rational chemical design used in the chemistry of aromatics, and taking into consideration availability of the substrates, inductive effects, efficiency ofthe chemical steps, and the cost ofthe reagents.
• Possible labeling methods are not limited to the ring of the isothiocyanate.
• cyclized 2-anilino-5-thiazolinone (ATZ) amino acid is reacted with primary amino group of labeling reagent e.g. [ 125 I]-iodohistamine or 4-aminofluorescein (Tsugita et.al. 1988, and 1989).
• labeling reagent e.g. [ 125 I]-iodohistamine or 4-aminofluorescein (Tsugita et.al. 1988, and 1989).
• the amine protecting group can be modified, and the use of monomethoxytrityl (4-methoxy-triphenyl methyl, MMT) is considered.
• MMT monomethoxytrityl
• An alternative for the above protocol is the use of B AMPITC in the sequenator, and labeling prior to HPLC. This procedure involves an additional step of 4-aminomethyl reaction preferably with Bolton-Hunter reagent (radioiodinated derivative, see Figure 8), and subsequent HPLC analysis.
• a third method is the reaction of 2-iniline-5-thiazolinone (ATZ) amino acid with labeled primary alifatic amines, preferably attached to heterocyclic aromatic ring.
• AZA 2-iniline-5-thiazolinone
• [ I]-iodohistamine is used.
• Chromatography under control of MPD instrumentation can be used to minimize impurities to the attomole level.
• the criteria for selecting a promising reagent are:
• the reagent should be available following simple synthesis routes which allow it to be purified easily to the highest possible grade. 2.
• the reagent should be volatile. This permits the removal of the excess reagent after the reaction without losses of peptides or proteins. PITC and methylisothiocyanate (MITC) are preferred in this regard.
• the coupling of the reagent to the free amino groups of the proteins should exceed 90%; the reaction must be as complete for hydrophilic and small residues as for those with bulky and hydrophobic side chains. This is only possible if the reactive group of the reagent is not sterically hindered.
• the reagent should provide a radiophore prior to or after the degradation, which enables sensitive detection ofthe released amino acid derivatives. If the radiophore is to be provided after degradation, the reagent should have a protected group for binding the radiophore which is deprotected after coupling to the amino acid.
• Edman-type reagents according to the invention meet all these requirements. Many different isothiocyanate-type reagents are compatible with I radioderivatization. Only the classical PITC reagent has been studied in much detail, but synthesis of other suitable radiolabeled isothiocyanate homologues can be accomplished by persons of ordinary skill.
• radiodinated dansyl-PITC (DNSAPITC) is suitable for sequencing; the reagent produces PTH-amino acid derivatives which are detectable in low picomole quantities employing HPLC separation and detection with a fluorescence detector.
• the application os small-sized polyamide thin-layer sheets for additional identification of 1 to 10 pmoles has been demonstrated. This allows safe microsequencing of high reliability.
• the excellent stability of the derivativess add to the quality of this Edman-type reagent.
• manual sequencing employing DNSAPITC can be performed on the 50-100 attomole level and with many samples at the same time. Hence this reagent serves as a possible alternative to the classical PITC or DABITC/PITC sequencing approaches.
• lanthanide complexes can be used in the chemistry of the Edman degradation. It has been shown that lanthanide complexes are sufficiently stable to be used for HPLC (Okabayashi et al., 1994).
• Chelating agents for lanthanides include N-benzyl diethylenetriaminetetraacetic acid.
• the chelator is N-(p- isothiocyanatobenzyl) diethylenetriaminetetraacetic acid.
• Eu +3 chelate fluorescence detection is known to have many limitations, especially in HPLC, where e.g. increase of acetonitrile concentration above 20% causes rapidquenching of fluorescence. Quenching introduces significant errors; thus fluorescence based mehtods are not quantitative.
• the innovative substitution of a radiolanthanide for fluorescent Eu comples avoids problems of fluorescence quenching which have been observed in prior art (Mukkala et al., 1989).
• Other chemically campatible chelates can be used, for example trisbipyridine cryptates (Lopez et al., 1993). MPD-enhanced detection is fully quantitative, as radioactivity is independent of chemical or physical factors.
• HP-TLC High-Performance thin layer chromatography
• RP-TLC Reversed Phase TLC
• CE-LIF Laser Induced Fluorescence detection
• An important feature of the present invention is its ability to provide a highly parallel sequencing process.
• modern molecular biology there is an increasing need for high- throughput analysis of proteins.
• Many very important proteins are present in minute amounts, because they provide very powerful signals for the transduction cascades.
• sensitivity of protein detection, and especially super-sensitive, high-throughput sequencing can significantly facilitate protein analysis.
• Embodiment of the invention can be used for parallel sequencing of many (ca. 50) proteins from 2D gels at the zeptomole level, where Edman degradation products can be separated and analyzed (detected and quantitated) simultaneously on a TLC plate.
• TLC plates are compatible with the MPD-imager format, where detection of the peaks is very rapid, and then it is possible to quantitate the peaks with increasing and adjustable spatial resolution. This approach has lower cost than CE-LIF or especially HPLC/MS.
• the efficiency of the chemical reactions is important. Because the amount of analyzed sample is so small, the reaction rate at the coupling step is diminished according to kinetics. It is therefore necessary to use the reagent in excess. Other steps (cleavage and conversion) are intramolecular and their rate is concentration independent. Acid catalysis, especially gas- phase, provides stoichiometric excess of the TFA, so appropriate protonation steps are not rate limiting.
• TLC is an advantageous separation medium for analysis of the Edman degradation products. Although this method was used with success in 1960s, the was a complete shift to HPLC and CE in automated sequenator design. However, use of High-Performance or Reversed Phase gels in TLC allows at least equal separation efficiency at lower cost and much higher throughput, and TLC readout is preferable for multi-photon detection than HPLC, because of the multi-photon detection-imager characteristics.
• Protein microsequencing may also be carried out with a modified HPLC readout, using MPD techniques.
• HPLC is used to fractionate the amino acids in "flow" mode.
• an appropriate optical detector UV absorbtion, fluorescence
• UV absorbtion, fluorescence is placed at the end ofthe column and the amount of reagent derivatized to increase the optical signal, either emission or absorbtion, is measured when it passes through the small size optical element.
• This implementation is not efficient when using 125 I as a label, because the time of transit through the active zone is very short, typically about 1% of total time of separation.
• the radioactive decay process is time-extensive, i.e.
• the time of transit of a given Edman degradation product through a 20 cm long HPLC column is 20 minutes and the fraction has a concentration of about 50 attomole, corresponding to 100 decays per minute (100 dpm).
• the separation ofthe Edman degradation product happens within the first part of the HPLC column, even if the optimal spatial resolution (fully formed, narrow peak) is obtained only at the column exit.
• the full width, half maximum (FWHM) of this peak is 1 cm at the distance of 10 cm and 0.5 cm at the column exit.
• the peak displacement speed can be reliably calculated from its movement in the first part of the column.
• a one dimensional MPD spacially resolving detector with aperture of about 2 cm and spatial resolution of 0.2 mm is placed at about the halfway point of the HPLC column.
• the count rate is sent to a computer which calculates the peak profile.
• the peak can be established within about 20 sec with a precision of about 5 mm.
• the peak displacement speed can be calculated with better than 10% precision.
• the SR-MPD device can be co-moved with the peak and during the next ten minutes a full profile of activity in the peak is acquired. For 50 attomole of Edman degradation product, the total amount of counts acquired is calculated to be about 1,000 which permits quantitation of amount of analyte with statistical precision better than 3%. Also, the peak position can be established with precision better than 1 mm.
• MPD detectors are virtually zero background devices, qualitative detection of minute amounts, e.g. only three counts, is statistically meaningful. That is, when using MPD one can very quickly establish that a given "pixel” or “voxel” contains I or another MPD- compatible label. However, quantitation may require a longer time. Typically one would like to acquire at least 100 counts/pixel to obtain 10% statistical uncertainty level.
• the diverse fractions of HPLC output are retained on the surface of appropriate filtering medium, e.g. a moving band of filter paper.
• appropriate filtering medium e.g. a moving band of filter paper.
• the length ofthe chromatogram is between half and twice the length ofthe HPLC column. First, all of the chromatogram is very quickly scanned by an MPD device for the presence of a peak. Next, a high precision but much slower quantitation process is performed.
• a MPD detector is equipped with changeable lead aperture and the object to be scanned, e.g. a one- dimensional chromatogram, is mechanically moved past the aperture.
• the full length ofthe chromatogram is 20 cm, i.e. about the same as the length of typical high resolution HPLC column.
• the width of chromatogram is arbitrary, but in a preferred implementation it is smaller than 1 cm.
• the MPD detector may have a diameter of 2.54 cm.
• the first step is to scan the chromatogram with the "wide aperture" with dimensions of 2 cm x 1 cm. Thus in ten steps the whole length of the chromatogram can be scanned.
• the position of the peak is established with a precision better than 0.5 mm.
• the activity profile of the peak is established with good precision, i.e. the number of counts under the peak is established with a precision better than 5%.
• a standard with retention time slightly lower than first Edman degradation product is loaded at a level of at least 100 femtomole into the column.
• HPLC apparatus with an in-flow optical detector may be used to detect the transit of the standard marker. Then, the pressure is released leading to a "frozen" pattern of the Edman degradation products inside the column.
• Such a column can then be scanned or imaged by an appropriate MPD detector. This is feasible because high energy X/gamma rays emitted by I easily cross the thin wells of the column, even if stainless-steel columns are used.
• the above-described strategy of repeated interactive MPD scans with three variable dimensions aperture can also be implemented in this case.
• the column typically has less than 2 mm diameter as compared with a 1 cm chromatogram as described above, appropriate apertures are 2 cm x 0.5 cm, 0.5 x 0.5 cm and 0.2cm x 0.5 cm, respectively.
• the quantitation process will typically take from a few minutes to about 30 minutes for less than 20 attomole fractions. In this time, diffusion may lead to considerable smearing ofthe peak. Thus when measurement time is expected to be above a few minutes, the diffusion should be diminished by cooling the column, preferably below the freezing point ofthe buffer, or other means.
• the multicolumn HPLC system is used and the "freezed" pattern is obtained essentially simultaneously in all columns in parallel.
• the columns can not be mechanically removed and placed onto an appropriate 2D MPD Imager.
• an SR MPD imager (4 columns x 10 detectors per column) is possible using a 2 inch SR MPD. This arrangement leads to about 20 fold faster read-out than when using a single detector. Multiple crystals read by a photodiode array can achieve a further 10 fold acceleration ofthe quantitation process.
• a supersensitive MPD-enhanced quantitation process according to the invention starts with
• the peak are well resolved and their position is well known a priori.
• the dynamic range of peaks amplitude is limited, typically less than 1 log. Actually, when the products of each cycle are fractioned separately there is only one peak and for all cycles the amplitude is the same.
• the amplitudes of different peaks after normalization to the highest peak generally give a characteristic pattern of fractional numbers for amplitudes, i.e. amplitudes are 1, 1/2, 1/3, 1/4...1/i wherein "i" is integer number.
• amplitudes are 1, 1/2, 1/3, 1/4...1/i wherein "i" is integer number.
• the end part of the HPLC column is shaped with a constriction ofthe appropriate size.
• the flowing liquid is accelerated and can be induced to break into droplets of very small, submillimeter diameter.
• the process of creating drops is accelerated by applying to the end part of the HPLC column vibrations of appropriate amplitude and frequency.
• high frequency pressure can be applied through the HPLC itself, i.e. the constant or gradient pressure operation of HPLC is modulated by a high frequency but relatively low pressure component.
• the end part of the HPLC capillary is made of an elastic material, e.g. plastic tubing whose geometrical dimensions are modulated by an external actuator.
• the said actuator can be either mechanical, electromagnetic or piezoelectric.
• the HPLC column end is placed into a high pressure gas chamber in which the pressure is modulated leading to an oscillating differential pressure which induces droplet creation.
• the pressure in the "gas chamber” from vacuum to atmospheric pressure, the creation of droplets can be effected.
• the selected HPLC conditions may involve the use of a pressure gradient leading to time variable flow speed.
• the diameter and number of created droplets may be time dependent.
• the drops are collected on the moving band consisting of the absorbent material.
• the properties of the material e.g. porosity, are selected so that all amino acids are retained but the HPLC buffer moves freely across the band of the moving material.
• the absorbent material may be divided into millimeter size pixels of absorbent material characterized by high diffusivity, and submillimetric walls made of material with substantially lower diffusivity.
• the size of the pixels is 0.5 to 5 mm and the walls are less than 0.2 mm thick.
• the preferred implementation of such a pattern is by controlled spraying of nonporous plastic upon a filter paper band.
• a chromatogram can be interpreted as a sequence of 0 or 1 bits, and subsequent quantitation is considerably accelerated, i.e. only the domains which absorbed the droplet are quantitated.
• the appropriate information can be stored in a computer to be subsequently used for optimizing the scheme of detection using an MPD Imager.
• the buffer of HPLC can be tinted with an appropriate colorant.
• 19S moving band can be read optically by the appropriate optical detector and the I content of only colored pixels is measured by an MPD Imager.
• the chromatogram may be essentially two-dimensional and the mechanical system consisting of a computer steered x-y mover with at least 100 microns precision to permit moving the absorbing material in a zig-zag pattern similar to TV raster pattern. This produces a 2D array of pixels.
• the x-y mover is operated to obtain a spiral pattern of droplets adsorbed on the surface of a chromatogram.
• CCD-based MPD imagers it may be possible to quantitate concurrently all pixels of 128x128 a pixels chromatogram.
• TLC based implementations Two-dimensional TLC ofthe PTH-amino acid products has been used in protein sequencing. Modern silica gels allow achieving resolution capabilities comparable to HPLC systems. Two TLC gel types high performance (HP TLC), and reversed phase (RT TLC) are fully compatible with MPD techniques. Resolution, sensitivity and reproducibility of MPD-enhanced TLC has been previously shown. HP TLC can be developed in a gradient of pH, while RT TLC can use a gradient of organic solvent (typically acetonitrile).
• HP TLC can be developed in a gradient of pH
• RT TLC can use a gradient of organic solvent (typically acetonitrile).
• An advantage ofthe proposed implementation is an increase in the throughput ofthe parallel sequencing reactions.
• Single TLC can easily accommodate 50 samples that can be the products of 50 simultaneously performed sequencing operations on individual blot pieces cut out of the two- dimensional gel. At the end of each cycle the products are separated on separate TLC plate and analyzed by MPD imager.
• the increase in throughput compensates more than enough for the manual handling necessary in the simplest prototype versions.
• the cost of TLC-based analysis is very moderate in comparison with HPLC equipment. Automation brings further advantages.
• the embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. None in this specification should be considered as limiting the scope of the present invention.
• Hunkapiller RM Hood LE, Dreyer WJ (1981): A gas liquid solid phase peptide and protein sequenator. J Biol Chem 256:7990.
• Mc Garvey B.D. Derivatization reactions applicable to pesticide determination by high- performance liquid chromatography. J. Chromatogr., B, 659, 243-257 (1994). Moore S, Stein WH (1963): Chromatographic determination of amino acids by the use of automatic recording equipment. Colowich SP, Kaplan NO (eds): "Methods in Enzymology.”
• Timashoff SN (eds): "Methods in Enzymology.” New York: Academic Press, Vol 27, pp 942-1010.

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