WO2000012994A1 - Apparatus for rapid protein and polypeptide sequence analysis - Google Patents

Apparatus for rapid protein and polypeptide sequence analysis Download PDF

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Publication number
WO2000012994A1
WO2000012994A1 PCT/US1999/019460 US9919460W WO0012994A1 WO 2000012994 A1 WO2000012994 A1 WO 2000012994A1 US 9919460 W US9919460 W US 9919460W WO 0012994 A1 WO0012994 A1 WO 0012994A1
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WO
WIPO (PCT)
Prior art keywords
cartridge
sample
sequencer
port
reagent
Prior art date
Application number
PCT/US1999/019460
Other languages
French (fr)
Other versions
WO2000012994A9 (en
Inventor
William J. Henzel
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Genentech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU56926/99A priority Critical patent/AU5692699A/en
Publication of WO2000012994A1 publication Critical patent/WO2000012994A1/en
Publication of WO2000012994A9 publication Critical patent/WO2000012994A9/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
    • G01N35/1097—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers characterised by the valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/025—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having a carousel or turntable for reaction cells or cuvettes

Definitions

  • Provisional Application Serial No. 60/098,401 filed on August 31 , 1998.
  • the contents of Provisional Application Serial No. 60/098,401 are inco ⁇ orated herein by reference.
  • the invention relates to apparatus and methods for protein or polypeptide sequencing.
  • the invention relates to devices using continuous flow chambers for protein or polypeptide sequencing, and to methods for protein or polypeptide sequencing which use flow through chambers.
  • Edman Degradation Method is based on the reaction between an amino group and phenyl isothiocyante to form a substituted thiourea. Mild hydrolysis with acid selectively removes the N-terminal residue of a polypeptide as the phenylthiohydantoin. Thus, the degradation is a cyclic procedure, by which an amino acid residue is cleaved one at a time from the N-terminus of the polypeptide and identified as the phenylthiohydantoin derivative.
  • the first step is coupling of phenylisothiocyanate (PITC) with the amino-terminal residue.
  • the second step is cleavage of the amino-terminal residue via cyclization in acidic medium.
  • the third step is conversion of the thiazolinone (ATZ) derivative formed during cleavage to generate the more stable thlohydantoin (PTH) derivative, which may be identified chromatographically (Burdon, R.H., ed., "Sequencing of Proteins and Peptides," in Laboratory Techniques in Biochemistry and Molecular Biology, vol. 9, chpt. 6, pp. 221-230 (1993)).
  • Automated sequencing has greatly enhanced the speed with which proteins and polypeptides may be identified.
  • Automated liquid phase protein and polypeptide sequencing began in 1967 with the introduction of the liquid phase spinning cup sequenator in which the tagging and cleavage reactions occur in a thin liquid film formed on the inside wall of a spinning reaction vessel. (Edman, P. and Begg, G., European J. of Biochem. 1:80-91 (1967).
  • sample loss particularly for short polypeptides, was a problem with this method.
  • the protein or polypeptide of interest may be trapped within a solid matrix or immobilized on a solid support matrix, such as a glass fiber matrix, a macoporous polystyrene matrix, or glass beads, by covalent attachment or by adsorption.
  • a solid support matrix such as a glass fiber matrix, a macoporous polystyrene matrix, or glass beads.
  • the reagents and solvents are passed through a column packed with the solid matrix to which the polypeptide is attached.
  • the protein or polypeptide is blotted onto a polyvinylidene difluoride (PDVF) membrane following 2-D gel electrophoresis and the portion of the membrane to which the protein or polypeptide is attached is analyzed by the solid phase Edman Degradation Method.
  • PDVF polyvinylidene difluoride
  • Gooley et al. An alternate method was described by Gooley et al. (Gooley, A.A. et al., 30 Electrophoresis 18:1068-1072 (1997)). To avoid the extended time necessary for sequencing 10 amino acid residues, Gooley et al. sequenced only 3 - 5 residues of each sample. Because protein identification cannot be done with so little sequence data, an amino acid analysis was also performed on each sample. The combination of on-line sequencing analysis with off-line amino acid analysis was required to confidently identify sample proteins. It was also proposed in Gooley et al. that other off-line techniques such as polypeptide masses, apparent protein mass (predicted by SDS-PAGE) and protein pi may be combined with 3 - 5 residue N-terminal sequence information to identify proteins. However, such mixed on-line and off-line analyses are cumbersome, time-consuming, and ultimately, more costly for the user.
  • the apparatus includes a cartridge holder, a plurality of apparatus sample cartridges and a fluid connector.
  • Each apparatus sample cartridge defines a chamber which receives one of the samples.
  • Each apparatus sample cartridge is formed with a cartridge inlet and a cartridge outlet so that fluid can flow through the apparatus sample cartridge.
  • the fluid connector selectively and independently connects a reagent port of a reagent delivery device in fluid communication with the cartridge inlet of each apparatus sample cartridge. This allows a single reagent delivery device to supply chemicals, i.e., reagents or solvents, to a plurality of apparatus sample cartridges. This reduces the cost to manufacture and operate the apparatus.
  • the fluid connector can be a multi-port radial valve having a connector inlet port, a connector outlet port, a plurality of supply ports and a plurality of return ports.
  • the connector inlet port is attached in fluid communication with the reagent port of the reagent delivery device.
  • Each apparatus sample cartridge is connected to one of the supply ports.
  • the cartridge inlet of each apparatus sample cartridge is connected in fluid communication with one of the supply ports of the valve.
  • the fluid connector also provides a path of fluid communication from the cartridge outlet of each apparatus sample cartridge to a conversion chamber and then to an analyzer. This allows a residue created by the interaction between the reagent and the sample to be transferred to the analyzer for analysis.
  • the cartridge outlet of each apparatus sample cartridge is connected in fluid communication with one of the return ports of the valve.
  • the connector outlet port of the valve is connected in fluid communication with the conversion chamber and an analyzer port. In this manner, an independent path of fluid communication can be established from the cartridge outlet, through one of the return ports, through the valve outlet port and into the analyzer port. This allows a single analyzer to separately analyze the residue from multiple apparatus sample cartridges.
  • the cartridge holder retains the plurality of apparatus sample cartridges.
  • the cartridge holder can be a printed circuit board having a plurality of holding stations. Each holding station is formed with a channel which is sized and shaped to receive one of the apparatus sample cartridges oriented substantially horizontal. Each holding station can also include a heater. The heater allows for individual heating of each individual apparatus sample cartridge to a desired temperature, from about ambient temperature to about 90 °C, inclusive.
  • the apparatus can be arranged as a module which is adapted for use with an existing sequencer having a plurality of sequencer sample cartridges. More specifically, the module includes a module base which retains the cartridge holder, the plurality of apparatus sample cartridges and the fluid connector as described above. Preferably, the module would include at least two and more specifically, at least five apparatus sample cartridges. In this embodiment, the module is connected to the sequencer in the place of one of the sequencer sample cartridges. Thus, the module expands the number of sample cartridges accessed by the sequencer and increases the throughput of the sequencer. Additionally, as provided herein, more than one module can be added to the sequencer to further expand the throughput of the sequencer.
  • each holding station includes an inlet tube which is in fluid communication with one of the cartridge inlets and an outlet tube which is in fluid communication with one of the cartridge outlets. Further, the reagent outtake and the analyzer intake are secured to a port holder.
  • the reagent outtake is in fluid communication with the reagent port and the reagent delivery device while the analyzer intake is in fluid communication with the analyzer port and the analyzer.
  • the cartridge holder is moved to selectively align the inlet tube and the outlet tube of one holding station with the reagent outtake and the analyzer intake, respectively.
  • reagent can be individually delivered to each sample cartridge from a single reagent delivery device by selectively moving the cartridge holder. Further, the residue from the sample cartridges can be individually transferred to the analyzer for analysis.
  • a sample is placed in the chamber of one of the apparatus sample cartridges.
  • One or more reagents are selectively delivered to each apparatus sample cartridge. Residue from each apparatus sample cartridge is independently delivered to the analyzer.
  • the operation of the apparatus provided herein is preferably done according to a rapid, approximately twenty minute (20 min) Edman cycle as provided below. In this manner, a plurality of samples are rapidly and accurately sequenced by the apparatus of the present invention.
  • Figure 1 A is a top plan illustration of an apparatus having features of the present invention
  • Figure IB is a top plan illustration of an apparatus and a sequencer having features of the present invention
  • Figure 1C is a top plan illustration of a plurality of apparatuses and a sequencer having features of the present invention
  • Figure 2 A is a perspective view of a holding station and a sample cartridge having features of the present invention
  • Figure 2B is a cross-sectional view of a sample cartridge, a sample, a portion of a supply line and a portion of a return line having features of the present invention
  • Figure 3 is a perspective view of an alternate embodiment of an apparatus having features of the present invention.
  • Figure 4 is a cross-sectional view taken on line 4-4 in Fig. 3;
  • Figures 5A-5L are chromatographic traces of PTH-amino acids following a sequence analysis pursuant to the present invention of mouse insulin-like growth factor binding protein 4 electroblotted onto PVDF;
  • Figures 6A-6E are chromatographic traces of the PTH-amino acid standards separations using different chromatograph conditions in conjunction with the apparatus of the present invention;
  • Figures 7A and 7B are histograms illustrating an average amino acid yield of three analyses of PVDF electroblotted myoglobin (Fig. 7A) and beta-lactoglobulin (Fig. 7B) using the 20 minute Edman cycle provided herein, the single letter designations along the x-axis represent the amino acids detected;
  • Figures 8A-8K are chromatographic traces of PTH-amino acids following sequence analysis of a 10 pmol sample of myoglobin electroblotted onto PVDF using a rapid Edman cycle described herein applied to 10 consecutive residues of the protein, the peak labeled " W" is an oxidized derivative of tryptophan; and
  • Figure 9 is a bar graph which highlights the repetitive yield accomplished using the apparatus and methods of the present invention on three separate protein samples.
  • FIG. 1A illustrates a first embodiment of an apparatus 10, for rapid protein sequence analysis of multiple samples 12 having features of the present invention.
  • the apparatus 1A includes a reagent delivery device 14, a fluid connector 16, a conversion chamber or conversion chamber 17, an analyzer 18, a plurality of apparatus sample cartridges 20, a cartridge holder 22, and an apparatus controller 24.
  • the apparatus 10 is useful for selectively and individually providing a plurality of chemicals, i.e., solvents or reagents 30 from a single reagent delivery device 14 to the plurality of sample cartridges 20. Subsequently, a residue 32 from each apparatus sample cartridge 20 is individually transferred to the conversion chamber 17 and then to the analyzer 18 for analysis.
  • the apparatus 10 provided herein is particularly useful for the identification of an amino acid residue 32 released from the sample 12 following the coupling, cyclization, and conversion reactions in the individual, apparatus sample cartridges 20.
  • each sample 12 is typically a protein or a polypeptide of unknown structure and biological function.
  • Each protein or polypeptide can be blotted onto a polyvinylidene fluoride (PVDF) membrane following 2-D gel electrophoresis. Subsequently, a portion of the membrane containing the sample 12 can be placed within one of the apparatus sample cartridges 20.
  • PVDF polyvinylidene fluoride
  • Each sample 12 can be absorbed or covalently attached to the membrane so that the protein or polypeptide is not dislodged during the coupling and cyclization reactions.
  • the protein or polypeptide of interest may have been isolated from other proteins or polypeptides by gel electrophoresis and a portion of the gel containing the protein or polypeptide is cut from the gel and transferred to the membrane.
  • the reagent delivery device 14 delivers a predetermined volume of one or more selected solvents and reagents 30 to the apparatus sample cartridges 20.
  • suitable reagent delivery devices 14 include, but are not limited to, HEWLETT-PACKARD Model 61005TM and PROCISE 494TM and PROCISE 473TM sold by Applied Biosystems of PE Biosystems located in Foster City, California.
  • the reagent delivery device 14 illustrated in Figure 1A includes a single reagent port 36 which delivers the solvents and reagents 30. Suitable reagents and solvents 30 are outlined in the examples provided below.
  • the conversion chamber 17 initially receives the residue 32 from each sample cartridge 20.
  • the conversion chamber 17 is also referred to as a conversion flask.
  • the conversion chamber 17 is preferably a container which is lined or made of a chemically inert material such as polytetraflouroethylene or glass.
  • a suitable polytetraflouroethylene is sold under the trademark TeflonTM.
  • the conversion chamber 17 preferably includes a heater (not shown) which heats the conversion chamber 17 to between approximately ambient to 70°C, inclusive.
  • reagents 30 and residues 32 in which the thiazolinone (ATZ) derivative formed during cleavage of the amino terminal residue is converted to the detectable more stable thiohydantoin (PTH) derivative.
  • the conversion chamber 17 is downstream of the sample cartridge 10 and upstream of the analyzer 18.
  • the analyzer 18 includes an analyzer port 38 for receiving the residue 32 from the conversion chamber 17.
  • the analyzer 18 can include, but is not limited to, a gas or high pressure liquid chromatographic column 40, and/or an amino acid derivative detection system 42 (such as a light, mass, charge or other physical detection technique available at the time of analysis).
  • the amino acid derivative detection system 42 can preferably detect an amino acid derivatized and released from the sample 12 of interest with sufficient sensitivity to reproducibly identify a given amino acid above the background signal.
  • the fluid connector 16 individually and independently connects each of the apparatus sample cartridges 20 to the reagent port 36 and the analyzer port 38 via the conversion chamber 17.
  • the fluid connector 16 is a multi-port, multi-position, radial valve 43 which includes a connector inlet port 44 which is in fluid communication with the reagent port 36. More specifically, in the embodiment illustrated in Figure 1A, a reagent tube 46 connects the reagent port 36 in fluid communication with connector inlet port 44.
  • the valve 43 illustrated in Figure 1A also includes six supply ports 48.
  • the supply ports 48 provide individual and independent paths for solvents or reagents 30 to flow from the connector inlet port 44 to each of the apparatus sample cartridges 20.
  • a plurality of supply tubes 50 are used so that each supply port 48 is in fluid communication with one of the sample cartridges 20. More specifically, a separate supply tube 50 is used to connect one of the supply ports 48 to one of the apparatus sample cartridges 20. Stated another way, one of the supply tubes 50 extends between one of the supply ports 48 and a cartridge inlet 52 to one of the apparatus sample cartridges 20. This allows solvents and reagents 30 from the reagent delivery device 44 to be independently delivered to each individual apparatus sample cartridge 20.
  • the valve 43 also includes a connector outlet port 54 in fluid communication with the analyzer port 38 via the conversion chamber 17. More specifically, in the embodiment illustrated in Figure 1A, an analyzer tube 56 connects the analyzer port 38 in fluid communication with connector outlet port 54 via the conversion chamber 17.
  • the valve 43 illustrated in Figure 1A also includes six return ports 58.
  • the return ports 58 provide individual and independent paths for residue 32 to flow from each apparatus sample cartridge 20 to the connector outlet port 54.
  • a plurality of return tubes 60 are used so that each return port 58 is in fluid communication with one of the apparatus sample cartridges 20. More specifically, a separate return tube 60 is used to connect a cartridge outlet 62 of one of the sample cartridges 20 to one of the return ports 58. Stated another way, one of the return tubes 60 extends between one of the cartridge outlets 62 and one of the return ports 58. This allows the residues 32 from each sample cartridges 20 to be delivered independently to the conversion chamber 17 and then the analyzer 18.
  • the valve 43 is selectively controlled to establish an individual and independent path of fluid communication from the reagent delivery device 14, through the connector inlet port 44, through one of the supply ports 48, and to one of the sample cartridges 20. Somewhat similarly, the valve 43 is also selectively controlled to establish an individual and independent path of fluid communication from one of the apparatus sample cartridges 20, through one of the return ports 58, through the connector outlet port 54, to the conversion chamber 17 and to the analyzer 18.
  • the supply ports 48 and the return ports 58 of the valve 43 are equally spaced apart and oriented radially around the body of the valve 43. With this design, the fluid path from the connector inlet port 44 to each of the supply ports 48 is approximately the same length.
  • a suitable valve 43 for the embodiment illustrated in Figure 1 A is a 14-port stainless steel automated valve, sold by Valco Instruments Co., Inc., located in Houston, Texas under the model number EMT-CST-UWTF.
  • the reagent tube 46, the supply tubes 50, the return tubes 60, and the analyzer tube 56 are preferably made of a chemically inert material such as polytetrafluoroethylene.
  • a suitable polytetraflouroethylene material is sold under the trademark TeflonTM.
  • TeflonTM a suitable polytetraflouroethylene material is sold under the trademark TeflonTM.
  • Suitable tubing has an inner diameter of between approximately 0.3mm and 0.5 mm and an outer diameter of approximately 1/16 inch.
  • the apparatus sample cartridges 20 retain the plurality of samples 12 during sequencing.
  • Each apparatus sample cartridge 20 includes a chamber 64 which is sized and shaped to receive one of the samples 12.
  • Each apparatus sample cartridge 20 also includes the cartridge inlet 52 and the cartridge outlet 62 to allow for fluid flow through the chamber 64.
  • each cartridge inlet 52 is connected directly to one of the supply tubes 50 and each cartridge outlet 62 is connected directly to one of the return tubes 60 for quick and easy attachment and detachment of the apparatus sample cartridges 20.
  • the attachments between (i) the supply tube 50 and the apparatus sample cartridge 20 and (ii) the return tube 60 and the apparatus sample cartridge 20 preferably form a liquid-tight and gas-tight seal so that the sample 12, the reagents 30 and residue 32 are not appreciably lost from the apparatus sample cartridge 20.
  • each apparatus sample cartridge 20 is also quickly and easily inserted into or removed from the cartridge holder 22 to facilitate sample 12 changes.
  • the apparatus samples cartridges 20 are preferably made of a chemically inert material, such as glass or polytetraflouroethylene, sold under the trademark TeflonTM.
  • each apparatus sample cartridge 20 can be a hollow tube which receives the membrane and sample 12. Further, each apparatus sample cartridge 20 can have an outer diameter of approximately 1/8 inch and an inner diameter of approximately 1/16 inch. As illustrated in Figure 2B, this sizing allows the 1/16 inch outer diameter supply tube 50 and return tube 60 to fit tightly within the apparatus sample cartridge 20 and form a liquid-tight and gas-tight seal. Further, this arrangement allows the supply tube 60 and the return tube 50 to retain the sample 12 within the apparatus sample cartridge 20. Each apparatus sample cartridge 20 can be between approximately 15- 20 mm long.
  • the cartridge holder 22 retains the plurality of apparatus sample cartridges 20 spaced apart.
  • the cartridge holder 22 illustrated in Figure 1A includes a holder body 66 and a plurality of spaced apart holding stations 68.
  • the holder body 66 is a flat, planer shaped circuit board. Alternate embodiments of the holder body 66 can be utilized.
  • Each holding station 68 is adapted to retain one of the apparatus sample cartridges 20.
  • each holding station 68 can include a generally rectangular shaped holding block 70, formed with a channel 72 which is sized and shaped to retain one of the apparatus sample cartridges 20 press fit into the channel 72. Alternately, those skilled in the art will recognize other ways to secure each apparatus sample cartridge 20 to one of the holding blocks 70.
  • each holding station 68 includes a heater 74 for individually heating each apparatus sample cartridge 20 to expedite the reactions within the sample cartridges 20.
  • the heater 74 transfers heat to each holding station 68 surrounding the apparatus sample cartridge 20.
  • the heater 74 preferably heats the sample 12 to a range of from approximately ambient temperature to approximately 90°C, inclusive.
  • a suitable heater 74 can be a thermofoil strip attached in thermal communication to each holding block 70.
  • a suitable thermofoil strip is the model HK913L sold by Minco, located in Minneapolis, Minnesota.
  • each holding block 70 is made of aluminum. The aluminum provides a heat sink to maintain uniform temperature around the sample cartridge 20.
  • Each heater 74 of each holding block 70 is preferably designed to be individually activated and individually controlled by current from a source of electrical current 76.
  • each heater 74 (shown in Figure 2 A) is individually in electrical communication with the source of electrical current 76 with a separate electrical line (not shown).
  • the separate electrical lines are positioned side-by-side and form a bundle of electrical lines 78 illustrated in Figure 1A.
  • current may flow from the source of electrical current 76 through each electrical line to each individual heater 74 so that each heater 74 increases the temperature of the apparatus sample cartridge 20 to a desired temperature.
  • each holding station 68 can include an independent thermocouple sensor 75 (shown in Figure 2A) which measures the temperature of the holding station 68 and/or the apparatus sample cartridge 20. Information from each individual thermocouple sensor 75 can independently transferred to the apparatus controller 24 (shown in Figure 1A). With information regarding the temperature, the apparatus controller 24 can control the source of electrical current 76 so that each heater 74 heats the apparatus sample cartridge 20 to the desired temperature and at a desired time in the reaction cycle. Each heater 74 is preferably individually controlled by the apparatus controller 24. As provided herein, the apparatus controller 24 can control each heater 74 so that the apparatus sample cartridges 20 are all at the same or different temperatures.
  • the apparatus sample cartridges 20 are preferrably maintained in a horizontal orientation 80 by the cartridge holder 22 to minimize reagent evaporation and to allow maximum contact between reagents 30 and sample 12. Stated another way, the apparatus sample cartridges 20 are positioned so that a longitudinal axis 81 of each apparatus sample cartridge 20 is substantially horizontal.
  • the horizontal orientation 80 of each apparatus sample cartridge 20 provides superior sequence analysis results because of improved contact between the sample 12 and the reagents and solvents 30.
  • the horizontal orientation 80 of each apparatus sample cartridge 20 also enhances repetitive yield and improves sequence analysis accuracy.
  • the apparatus controller 24 is electronically connected to the fluid connector 16, i.e., the valve 43 with electrical line 82.
  • the controller 24 controls the position of the valve 43 so that (i) the solvents or reagents 30 are independently delivered to the appropriate apparatus sample cartridge 20, and/or (ii) residue 32 from the appropriate apparatus sample cartridge 20 is independently transferred to the conversion chamber 17 and then the analyzer 18.
  • the apparatus controller 24 can control a pneumatic and/or electrical motor 84 which moves the valve 43. Basically, the apparatus controller 24 controls the motor 84 so that the appropriate supply port 48 is independently in fluid communication with the connector inlet port 44 or the appropriate return port 58 is independently in fluid communication with the conversion chamber 17.
  • the fluid controller 24 controls the flow to and from each apparatus sample cartridge 20. Additionally, as provided above, the apparatus controller 24 is electrically connected to each holding station 68 so that the apparatus controller 24 can individually control each heater 74. Further, the apparatus controller 24 can include a display 85 which displays which sample 12 is being sequenced. In particular, the display 85 in Figure 1A includes a plurality of lights which illuminate to identify which sample 12 is being sequenced.
  • the apparatus 10 can include a plurality of optical sensors 86 for monitoring flow through the supply tubes 50 or the return tubes 60 to determine and/or detect whether there is fluid flow through each apparatus sample cartridges 20.
  • each optical sensor 86 monitors flow through one of the return tubes 60, near one of the apparatus sample cartridges 20. With this arrangement, each optical sensor 86 can monitor or detect when the chemicals 30 are positioned in one of the apparatus sample cartridges 20.
  • the optical sensors 86 illustrated in Figure 1A are electrically connected to a jack connection 88, which are electrically connected to the apparatus controller 24. Information from the optical sensors 86 can subsequently be individually transferred to the apparatus controller 24. This allows the apparatus controller 24 to monitor flow through each apparatus sample cartridge 20.
  • the embodiment illustrated in Figure 1A includes six apparatus sample cartridges 20 which sequence six separate samples 12. It is to be appreciated, however, that the apparatus 10 of Figure 1A can be designed to include more than six or less than six sample cartridges 20 provided the fluid connector 16 has a supply port 48 and return port 58 for each apparatus sample cartridge 20 or a second fluid connector (not shown) is utilized. Further, the cartridge holder 22 can include more than or less than six holding station 68.
  • Figure IB illustrates that the apparatus 10 can be arranged as a module 200 and used in conjunction with a commercial sequencer 202.
  • the module 200 illustrated in Figure IB includes a module base 204 which retains the various components of the apparatus 10. More specifically, the fluid connector 16, the apparatus sample cartridges 20, the cartridge holder 22, and the apparatus controller 24 are all secured to the module base 204.
  • the apparatus sample cartridges 20, the cartridge holder 22, the apparatus controller 24, and the fluid connector 16 are similar to that described above in the discussion of Figure 1A.
  • the apparatus 10 is arranged in this embodiment as a module 200 which can be added to the commercial sequencer 202.
  • six apparatus sample cartridges 20 are illustrated in Figure IB, the number of apparatus sample cartridges 20 for the module 200 can be more than six or less than six.
  • the sequencer 202 illustrated in Figure IB includes (i) a sequencer base 208, (ii) a sequencer reagent delivery device 210, (iii) a horizontally oriented, reagent port valve 212, (iv) four, sequencer sample cartridges 214, (v) a horizontally oriented analyzer port valve 216, (vi) a sequencer conversion chamber 218, (vii) a sequencer analyzer 220, and (viii) a sequencer controller 222.
  • the sequencer reagent delivery device 210 can be similar to the reagent delivery device 14 described above.
  • the sequencer conversion chamber 218 and the sequencer analyzer 220 respectively can be similar to the conversion chamber 17 and analyzer 18 described above.
  • one of the sequencer sample cartridges 214 is disconnected from the sequencer 202. Instead, the fluid connector 16 of the module 200 is connected in its place. Specifically, one of the reagent outlet ports 224 is connected (with line designated 46) in fluid communication with the connector inlet port 44 of the fluid connector 16 of the module 200. Somewhat similarly, the connector outlet port 54 of the module 200 is connected (with line 56) in fluid communication with one of the analyzer inlet ports 226 of the sequencer 202. Further, the apparatus controller 24 is electrically connected, via line 228 to the sequencer controller 222. This allows the sequencer controller 222 to control the fluid connector 16.
  • the module 200 is used to expand the throughput of the sequencer
  • the sequencer 202 illustrated initially utilized only four sequencer sample cartridges 214.
  • the module 200 having six apparatus sample cartridges 20, a total of nine samples 12 can be sequenced by the sequencer 202.
  • the module 200 can be used to increase the throughput of the sequencer 202.
  • FIG. 1C illustrates another embodiment of the present invention.
  • the commercial sequencer 202 is connected to four, separate individual modules 200.
  • Each module 200 is represented as a rectangle in Figure lC.
  • each module 200 illustrated in Figure 1C can be substantially similar to and include all of the components of the module 200 illustrated in Figure IB and described above.
  • each reagent outlet port 224 of the sequencer reagent delivery device 210 is connected to one of the modules 200, instead of the sequencer sample cartridges 214
  • each analyzer inlet port 226 is connected to one of the modules 200 instead of the sequencer sample cartridges 214
  • each module 200 is electrically connected to the sequencer controller 222.
  • Figure 3 illustrates another alternative embodiment of an apparatus 10 having features of the present invention.
  • the cartridge holder 20 also includes a plurality of spaced apart holding stations 68 for holding a plurality of spaced apart, apparatus sample cartridges 20.
  • a valve 43 (not shown in Figure 3) is not used to direct the particular reagents 30 from the reagent port 36 to each individual apparatus sample cartridge 20, and the residue 32 from each apparatus sample cartridge 20 to the conversion chamber 17.
  • a reagent outtake 87 and an analyzer intake 89 are secured to a port holder 90.
  • the reagent outtake 87 is in fluid communication with the reagent delivery device 14 and the analyzer intake 89 is in fluid communication with the analyzer 18 via the conversion chamber 17.
  • one of the holders i.e. the port holder 90 or the cartridge holder 22 is moved relative to the other holder so that the reagent outtake 87 and the analyzer intake 89 are selectively connected in fluid communication with one of the apparatus sample cartridges 20.
  • the cartridge holder 22 illustrated in Figure 3 is shaped similar to a carousel and includes twelve, spaced apart, holding stations 68. Further, in this embodiment, the cartridge holder 22 is selectively rotated about a central axis 92 by a mover 94 (shown in phantom in Figure 3). The mover 94 is mounted to the cartridge holder 22 below a bottom side 96 of the cartridge holder 22.
  • the cartridge holder 22 and apparatus sample cartridges 20 can be selectively moved by the mover 94 relative to the port holder 90.
  • the mover 94 can be a pneumatic and/or electrical motor or other means for moving the cartridge holder 22 relative to the port holder 90.
  • the mover 94 rotates the cartridge holder 22 to twelve alternate positions so that the twelve apparatus sample cartridges 20 are individually brought into fluid communication with the reagent outtake 87 and the analyzer intake 89.
  • linear movement of the cartridge holder 22 is capable of accomplishing the same result as the rotation of the cartridge holder 22 described herein, and is considered an embodiment of the invention.
  • the cartridge holder 22 can be designed to retain more than twelve or less than twelve apparatus sample cartridges 20.
  • the apparatus 10 could be designed so that the reagent outtake 87 and the analyzer intake 89 are moved relative to the cartridge holder 22 and the sample cartridges 20.
  • each holding station 68 includes a separate inlet tube 98 connected to the cartridge inlet 52 of the apparatus sample cartridge 20 and separate outlet tube 100 connected to the cartridge outlet 62 of the apparatus sample cartridge 20.
  • Each inlet tube 98 inserts through an inlet hole 102 in the cartridge holder 22 while each outlet tube 100 inserts through an outlet hole 104 in the cartridge holder 22.
  • Each inlet tube 98 includes an inlet tube distal end 106 which is secured to the cartridge holder 22 substantially flush with the bottom side 96 of the cartridge holder 22.
  • each outlet tube 100 includes an outlet tube distal end 108 which is secured to the cartridge holder 22 substantially flush with the bottom side 96 of the cartridge holder 22.
  • each inlet tube 98 and outlet tube 100 are preferably made of a chemically inert material, such as TeflonTM.
  • TeflonTM a chemically inert material
  • each inlet tube 98 and outlet tube 100 has an inner diameter of between approximately 0.3mm and 0.5 mm and an outer diameter of approximately 1/16 inch. However, those skilled in the art will recognize that alternate sizes can be utilized.
  • each holding station 68 can include a heater 74 and a thermocouple sensor 75 as described above.
  • the embodiment illustrated in Figure 3 also provides a unique way to electrically connect each heater 74 and thermocouple sensor 75 to the source of electrical current 76 and the apparatus controller 24.
  • each holding station 68 can include at least a pair of spaced apart heater electrical contacts 112a and a pair of spaced apart thermocouple electrical contacts 112b which are positioned on an outer perimeter 114 of the cartridge holder 22.
  • Each electrical contact 112a is electrically connected with electrical wires 116a to one of the heaters 74.
  • Each thermocouple electrical contact 112b is electrically connected with electrical wires 116b to one of the thermocouple sensors 75.
  • the source of electrical current 76 includes a resilient beam 1 18 which is urged against and contacts the outer perimeter 114 of the cartridge holder 22.
  • the beam 118 includes at least four beam electrical contacts 120 which are spaced apart.
  • Two of the beam electrical contacts 120 are adapted to contact the heater electrical contacts 112a of one of the heaters 34 and two of the beam electrical contacts 120 are adapted to contact the thermocouple electrical contacts 112b.
  • the beam electrical contacts 120 are electrically connected to the source of electrical current 76 and the apparatus controller 24 with source wires 122. This allows the apparatus controller 24 to monitor the temperature of each heater 74 and adjust the current to each heater 74 to adjust the temperature of each heater 74.
  • the beam 1 18 illustrated in Figure 3 includes vertical portion 124 and a transverse portion 126 which cantilevers away from the vertical portion 124 and contacts the outer perimeter 114 of the cartridge holder 22.
  • Figure 4 illustrates the interaction between the holding station 68, the reagent outtake 87 and the analyzer intake 89 in more detail.
  • the reagent outtake 87 and the analyzer intake 89 are mounted in a block shaped port holder 90.
  • the reagent outtake 87 includes a reagent outtake distal end 128 which is mounted substantially flush with an upper surface 130 of the port holder 90.
  • a reagent outtake seal 132 encircles the reagent outtake distal end 128 and extends away from the upper surface 130 of the port holder 90.
  • the analyzer intake 89 includes an analyzer outtake distal end 134 which is mounted substantially flush with the upper surface 130 of the port holder 90.
  • An analyzer intake seal 136 encircles the analyzer intake distal end 134 and extends away from the upper surface 130 of the port holder 90.
  • the reagent outtake distal end 128 and the analyzer intake distal end 134 are spaced apart substantially the same distance as the inlet tube distal end 106 and the outlet tube distal end 108.
  • the reagent outtake seal 132 interacts with the inlet tube seal 11 la to establish a fluid tight path of fluid communication between the reagent outtake 87 and the inlet tube 98.
  • the analyzer intake seal 136 interacts with the outlet tube seal 111b to provide a fluid tight path of fluid communication between the analyzer intake 89 and the outlet tube 100.
  • the inlet tube seal I l ia, the outlet tube seal 111b, the reagent outtake seal 132 and the analyzer intake seal 136 can each be an "O" ring type seal.
  • the cartridge holder 22 is moved by the mover 94 so that each sample cartridge 20 is individually aligned with the reagent outtake 87 and the analyzer intake 89, the electrical contacts 112a, 112b are aligned with the source electrical contacts 120.
  • Example 1 Automated Sequence Analysis Device. The chromatograms obtained from mouse insulin-like growth factor binding protein 4 (IGFBP-4), using the apparatus 10 of the invention are illustrated in Figs. 5A-5L.
  • IGFBP-4 mouse insulin-like growth factor binding protein 4
  • the partial N-terminal sequence of the protein was verified by the rapid Edman cycle method described herein to be DEAIHCPPCSE (SEQ ID NO:l) (Schuller A.G. et al. (1994) Mol. Cell. Endocrinol. 104:57-66, GenBank accession number 148600).
  • the device according to the present invention was used with rapid Edman cycles to sequence consecutive residues of the protein.
  • An Edman degradation cycle for protein sequencing typically involves coupling PITC with the amino terminal residue; cleavage of the amino terminal residue via cyclization in acidic medium; and conversion of the resultant ATZ derivative to a more stable PTH derivative that may be detected and identified.
  • the time required for one cycle of Edman degradation is dependent upon the time required to complete both the Edman chemistry and the PTH analysis.
  • the PTH chromatographic separation has been a primary limiting factor of cycle speed. Others have approached this problem by using higher flow rates at the expense of lower sensitivity (Totty et al. (1992) supra).
  • Chromatographic Figues 6A and 6B compare separations using a short column (0.2 cm x 15 cm, Fig. 6A) and a conventional chromatographic column (0..2 cm x 22 cm, Fig. 6B), each packed with 5 ⁇ m C18 resin.
  • the standard single letter designation for the amino acids are used to indicate the identity of the PTH-amino acid peaks. According to a method disclosed herein, increased speed, as well as increased sensitivity, are achieved through the use of a short chromatographic column (i.e., a 15 cm column instead of the standard 22 cm column). Figs.
  • 6C, 6D and 6E respectively compare separations using a 15 cm, 12.5 cm and 10 cm chromatographic column (each 0.2 cm diameter), respectively, packed with 3 ⁇ m C18 resin.
  • the time required to equilibrate the chromatographic column was also reduced by using a higher flow rate of 375 ⁇ l/min, rather than the standard flow rate of 325 ⁇ l/min of commercial devices, such as the PROCISE 140CTM, sold by Perkin Elmer- Applied Biosystems Division. Detection sensitivity increased because the short chromatographic column limited peak spreading.
  • the flow rate was programmed to be a specified rate at the beginning of each reaction cycle (which cycle includes coupling, cyclization and conversion of the N-terminal amino acid of the protein or polypeptide of interest).
  • the resultant flow gradient that occurs during the first minute of the separation as the flow rate rapidly decreases to the separation flow rate of 325 ⁇ l/min had no affect on retention time reproducibility of the derivatized amino acids.
  • the 15 cm chromatographic columns provided separations for an average of 2,000 to 3,000 injections before peak resolution began to decrease. Table 1 A lists chromatographic separation gradient conditions useful in the invention.
  • Solvent A of the gradient was 3.5% tetrahydrofuran (THF) in aqueous buffer comprising 2%
  • B may be 11-12.5% isopropanol in acetonifrile, where isopropanol may be useful to enhance separation of the PTH-tryptophan derivative from its oxidized form.
  • the target pressure is approximately 1500 psi and data collection time was approximately 20 min.
  • Solvent A of the gradient was 3.5% tetrahydrofuran (THF) in aqueous buffer comprising 2-4% Pre-MixTM buffer concentrate (Perkin-Elmer/Applied Biosystems). To each liter of Solvent A was added 15-25 ⁇ l acetone, 0.5 ml acetic acid, and 0.1 ml 1M Na2P04. For the 10 cm column, THF was 5% to resolve PTH-Gln and PTH-Thr. Solvent B was acetonifrile/ 11-12.5% isopropanol. Column temperature was 55°C.
  • Figs. 7A and 7B are histograms illustrating the average amino acid yield of three analysis of PVDF electroblotted myoglobin (Fig. 7A) and beta-lactoglobulin (Fig. 7B) using the 20 minute Edman cycle provided herein.
  • the single letter designations along the x-axis represent the amino acids detected.
  • Figs. 8A-8K are chromatographic traces of PTH-amino acids following sequence analysis of a 10 pmol sample of myoglobin electroblotted onto PVDF using a rapid Edman cycle as provided herein applied to 10 consecutive residues of the protein.
  • each Edman cycle as provided herein requires less than approximately 20 minutes, while the commercial method requires 33 minutes.
  • the temperature of the coupling and cleavage reactions of the present invention was increased from 48 °C to 55 °C, and the conversion temperature was increased to 75 °C.
  • Acetonifrile is a more efficient solvent for removing by-products from the conversion flask than a conventionally-used aqueous acetonifrile solution (Totty, N.F. et al., supra).
  • Fast Edman Cycle Method 1 is a more efficient solvent for removing by-products from the conversion flask than a conventionally-used aqueous acetonifrile solution (Totty, N.F. et al., supra).
  • Method 1 refers to a cycle of steps that may be used with reagent delivery devices for delivering a limited number of reagents or solvents to the sample cartridge and conversion flask 17 for reaction and wash steps.
  • R2 Methyl piperidine in n-propanol and water (25:60:15)
  • R5 PTH standard in acetonifrile (1 pmol/50 ⁇ l)
  • the "Begin,” “Flask Std.,” “Cartridge,” and “Flask Residue” cycles are overlapped in time to maximize efficiency and sample 12 throughput.
  • the Begin cycle may be run first on a first amino acid residue.
  • the Cartridge cycle is performed on the first amino acid residue, forming its ATZ derivative.
  • the Flask Std. is run as the Cartridge cycle is performed on the first amino acid residue.
  • the ATZ derivative of the first amino acid residue is transferred to the conversion flask 17 and converted to the PTH derivative at the same time that a second amino acid residue is undergoing the coupling reaction in the Cartridge cycle.
  • the cycles described herein and in Table 3 may be performed using a commercial protein sequencing reagent delivery device such as, but not limited to, a PROCISETM device, or its equivalent.
  • a commercial protein sequencing reagent delivery device such as, but not limited to, a PROCISETM device, or its equivalent.
  • the samples 12 are reacted in the sample cartridges 20 of the apparatus 10 of the present invention.
  • the apparatus 10 is operably coupled to the programmed Edman reaction cycles such that a different sample cartridge 20 is positioned for sample analysis upon completion of the predetermined number of amino acid residue analyses of the previous sample.
  • the "Begin” cycle (Table 3A) refers to an automated programmed cycle in which a protein sample is washed and a first coupling reaction is performed. This cycle is preferably performed once per sample as the beginning cycle to prepare the sample for further Edman cycles. More preferably, the Begin cycle is omitted since the coupling reactions of the "Cartridge” cycle are sufficient to achieve coupling of PITC to the N-terminal amino acid.
  • the Begin cycle is an optional feature and is disclosed in Table 3A. Steps 1-13 of the Begin cycle perform valve and block washings with heptane (solvent SI).
  • Steps 14-23 perform a sample wash in which the solvents such as, but not limited to, ethyl acetate (solvent S2), butyl chloride (solvent S3), and acetone (wash X3) are used.
  • Steps 24-38 perform the Edman coupling reaction in which PITC (PITC in heptane, reagent RI) is coupled to the N-terminal amino acid of the sample under basic conditions, such as in the presence of methyl piperidine in n- propanol and water (25:60: 15, designated reagent R2).
  • Steps 39-56 perform a sample and block wash using heptane (block wash) and ethyl acetate, butyl chloride, and acetone (sample wash).
  • the "Carfridge" cycle of Table 3B performs the coupling and cleavage reactions for each amino acid residue to be analyzed in the sample.
  • the steps in the Cartridge cycle are preferably performed at 55 °C.
  • Steps 1-16 perform the PITC coupling reaction by delivering 5% PITC in heptane (reagent RI, Table 3) to the sample in two portions under basic conditions in the presence of methyl piperidine in n-propanol and water (reagent R2, Table 3B).
  • Steps 17-33 perform a sample wash using ethyl acetate (S2), acetone (X3), and butyl chloride (S3).
  • Steps 34-48 perform cleavage and cyclization of the derivatized N-terminal amino acid to generate the ATZ-amino acid derivative under acidic aqueous conditions (such as in TFA (frifluoroacetic acid) in water, reagent R3, Table 3B).
  • Steps 49-68 transfer the ATZ derivative to the conversion flask.
  • the Flask Residue cycle of Table 3C is performed on a first amino acid derivative while the next amino acid residue in the sequence is coupled and cleaved in the Cartridge cycle.
  • the steps in the Flask Residue cycle are performed in a conversion flask 17 in-line between the sample carfridge and the chromatographic column, a typical configuration for a protein sequencing device.
  • the Flask Residue cycle is preferably performed at approximately 75 °C.
  • Steps 1-7 prepare the conversion flask 17 and receive the ATZ derivative from the Cartridge cycle.
  • Steps 8-19 convert the less thermodynamically stable ATZ-amino acid derivative to the more stable PTH-amino acid derivative under acidic conditions, such as in frifluoroacetic acid.
  • Steps 20-38 dry the conversion flask 17 and then dissolve the PTH derivative in 7.5% acetonifrile in water.
  • Steps 39-54 prepare the chromatographic injector and inject the PTH derivative onto an analysis system, such as a high pressure liquid chromatographic column for chromatographic analysis using the elution gradient shown in Table 1 A.
  • the user of the invention may perform an analysis of PTH-amino acid derivatives as necessary during the sample cycles.
  • a PTH-amino acid standard is run according to the Flask Std. cycle (Table 3D) performed simultaneously with the Begin cycle or the Carfridge cycle on the N- terminal amino acid of the sample.
  • the steps of the Flask Std. cycle are preferably performed at 75 °C.
  • Steps 1-5 of Table 3D wash the conversion flask with acetonifrile (X2, Table 3).
  • Steps 6-10 deliver the PTH amino acid standard (R5) to the flask.
  • Steps 11-20 allow the PTH amino acid standard to be exposed to the conditions of the conversion reaction.
  • Steps 21-38 dry the conversion flask and deliver acetonifrile to dissolve the PTH amino acid standard.
  • Steps 39-43 prepare the injector and inject the PTH amino acid standard onto the chromatographic column.
  • Steps 44-52 wash the flask and injector in preparation for the next cycle.
  • Method 2 provides steps useful in practicing Method 2 of Rapid Edman Cycles, which method is compatible with a reagent delivery system for delivering fewer reagents and solvents than a system useful for Method 1.
  • a regent delivery system useful in Method 2 may be, but is not limited to, a PROCISE 473TM reagent delivery system. As Table 4 shows, the Cartridge and Flask cycles are run simultaneously by a single program. The reagent and solvent designations used in Table 3 are also used in Table 4. Table 4. Method 2 of the Rapid Edman Cycle
  • R2 Methyl piperidine in n-propanol and water (25:60:15)
  • R5 PTH standard in acetonifrile (1 pmol/50 ⁇ l)
  • Steps 3-16 of Table 4 perform the PITC coupling reaction in the Carfridge cycle at 55 °C, while steps 1-19 perform the conversion of the ATZ-amino acid derivative to the PTH-amino acid derivative at 75 °C.
  • Step 20 directs the injector valve to be placed in the load position in the Cartridge cycle, while the chromatographic gradient is initialized by a command in the Conversion Flask cycle.
  • Steps 21-37 perform a solvent wash of the carfridge followed by drying, the conversion flask is also dried, and the injector is flushed and prepared to receive a converted amino acid derivative.
  • Steps 32-46 of the Conversion Flask cycle dissolve the PTH-amino acid derivative in 7.5% acetonifrile in water.
  • Steps 38-52 perform the cleavage reaction by delivering frifluoroacetic acid in gaseous form, thereby generating the ATZ-amino acid derivative.
  • Steps 47-49 in the Conversion Flask cycle load the injector with the PTH-amino acid derivative and inject it onto the chromatographic column, followed quickly by the start of the chromatographic solvent gradient.
  • Steps 53-73 of the Cartridge cycle dry the cartridge, extract the ATZ-amino acid derivative into ethyl acetate, and transfer the extracted derivative to the conversion flask.
  • Steps 53-73 of the Conversion Flask cycle clear and dry the conversion flask for receipt of the ATZ-amino acid derivative from the carfridge, and clear the injector following the injection in Step 48.
  • Table 1A describes the chromatographic analysis useful in the invention following the fast Edman cycles of Methods 1 and 2.
  • Example 3 Immobilization of Proteins onto PVDF Prior to Sequence Analysis.
  • This example describes a method of immobilizing proteins and polypeptides of interest onto a PVDF membrane prior to sequence analysis using the apparatus 10 of the invention with the rapid sequence analysis methods also provided herein.
  • Example 4 Automated Protein Sequencing.
  • the present example demonstrates use of the apparatus 10 of the invention in which the sample cartridges 20 are contained in a stationary sample cartridge holder 22 and the reagent 30 delivery is controlled with a multi-position valve 43 as illustrated in Figure 1.
  • a PROCISE 473 ATM reagent delivery device 14 was in communication with a stationary carfridge holder 22 which holds the sample cartridges 20 in the horizontal orientation 80.
  • Each sample cartridges 20 has a 1/16 inch internal diameter (i.d.) and a 1/8 inch outer diameter (o.d.).
  • the length of each sample carfridge 20 was approximately 15 - 20 mm.
  • the sample cartridges 20 were heated by a
  • carfridge selection was controlled by a 14-port stainless steel automated valve 43 (model EMT-CST6-UWTF, sold by Valco, Houston, TX).
  • the valve 43 was equipped with a multi-position actuator control module (Valco model EMTCA) which was controlled by an external relay in the PROCISE 473ATM protein sequencer.
  • Automated protein sequencing was performed on PE-Applied Biosystems protein reagent delivery devices, PROCISE 494ATM and a modified PROCISE 473ATM.
  • the PROCISETM reagent delivery device was used with a high pressure liquid chromatographic system.
  • the chromatography column 40 such as a 2.0 x 150 mm column by Alltech, (preferably PTH column part number C60003) was packed with 5 ⁇ m C18 resin (preferably PTH resin from Perkin Elmer- Applied Biosystems).
  • An on-line PTH analyzer such as a Perkin Elmer- Applied Biosystems 140CTM was also used.
  • the coupling buffer was N-methylpiperdine in 1-propanol and water (25:60:15).
  • solvent A comprised 3.5% THF in aqueous acetate buffer (such as 2% PremixTM Buffer Concentrate supplied by PE-Applied Biosystems).
  • Chromatographic solvent B (Table 1A) comprised approximately 11% (optionally 12.5%) isopropanol in acetonifrile (Burdick and Jackson, HPLC grade).
  • solvent B may be acetonifrile without added isopropanol.
  • Isopropanol was occasionally useful in enhancing the separation of the PTH-tryptophan derivative from phenylisothiocyanate.
  • acetone was routinely added to solvent A to balance the baseline. Peaks were integrated with Justice InnovationTM software using Nelson Analytical 760TM interfaces. Sequence interpretation was performed on a DEC AlphaTM computer according to the procedure described by Henzel et al. (Henzel, W.J. et al., J. Chromatogr. 404:41-52 (1987)).
  • the SEQSORT algorithm was used to sort sequence mixtures (W. J. Henzel et al., "Analysis of Mixture Sequences Derived From Edman Degradation Data,” in Techniques in Protein Chemistry, Marshak, D. R., ed., vol. VII, pp. 341-346 (1996)).
  • the algorithm finds patterns specified as regular- expression syntax. It is similar in implementation to the UNIX regular-expression matching program (see UNIX User's Reference Manual (URM), 4.3 Berkeley Software Distribution Virtual VAX-1 1 Version, Computer Systems Research Group, Department of Electrical Engineering and Computer Science, University of California, Berkeley, California).
  • ULM UNIX User's Reference Manual
  • SEQSORT has features beyond those of the UNIX regular-expression matching program.
  • SEQSORT features include (1) about 15 of allowed mismatches can be specified; (2) the search can be restricted to a region around the N- terminus; (3) the search can be limited to proteins with a specific molecular weight range; and (4) a species specific search can be performed.
  • the algorithm begins by compiling the ambiguous sequencer data using a finite automaton to find regular expressions.
  • the finite automaton described by Miller described by Miller (Miller, W., "A Software Tools Sampler,” Prentice-Hall, Englewood Cliffs, N.J. (1987)) was augmented by allowing transitions on mismatches, as long as the number of mismatches was below a user-specified threshold. Next, each sequence of the database was examined.
  • Sequences having a molecular weight outside the specified limits were rejected. If the search was restricted to a region near the N-terminus, the sequence was truncated to the region of interest. A default value of 60 residues was typically used to allow a match with proteins that contained signal sequences that were usually less than 40 amino acids in length. The resulting sequence is checked for the existence of the specified pattern. If the pattern is found, the sequence is added to a list which can later be sorted by molecular weight or by the number of mismatches. When no amino acid was observed on a cycle, an "X" was specified, allowing any amino acid to match. The algorithm sorted a mixture of over 100 sequences. It is noted that as the number of sequences increases, the number of random matches also increases, requiring longer sequence analyses to prevent random matching.
  • Example 6 Sequence Analysis of a Mixture of Proteins.
  • a proteolytically cleaved protein (most likely a mixture) was observed as a single spot on a 2- D gel and was analyzed to determine the sequence of 10 amino acids from the existing N-terminus.
  • a search of the protein sequence database, using this sequence of a potential mixture of proteins and the SEQSORT algorithm resulted in the identification of this sequence as intact apolipoprotein Cl and the cleaved form of this protein starting at residue 3.
  • the module 200 was connected to the sequencer 202 in place of one of the sequencer sample cartridges 214.
  • the module 200 was connected to the commercial sequencer, somewhat similar to that disclosed in Figure IB.
  • the module 200 effectively expanded a single sample position of the commercial sequencer 202 to five, sample positions. Expansion of each of the four single-sample positions of the sequencer 202 in this way expands the capacity of the commercial sequence 202 from four samples 12 to at least twenty samples 12. Further expansion is readily possible by increasing the number of apparatus sample cartridges 20 on each module 200. All references provided herein are hereby incorporated by reference in their entirety.

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Abstract

The invention relates to apparatus (10) for protein or polypeptide sample (12) sequencing. In particular, the apparatus (10) uses a plurality of continuous flow through sample cartridges (20). In one embodiment of the present invention, the apparatus (10) uses a valve (43) to direct one or more chemicals (30) from a single reagent delivery device (14) to the plurality of sample cartridges (20). Alternately, in another embodiment, the apparatus (10) moves the sample cartridges (20) relative to the reagent delivery device (14) so that a single reagent delivery device (14) again, can be used to supply chemicals (30) to a plurality of sample cartridges (20). The invention also uses a rapid, twenty minute, Edman cycle, coupled to a rapid and sensitive chromatographic separation of the PTH-amino acid derivatives so that sample (12) analysis throughput is maximized.

Description

APPARATUS FOR RAPID PROTEIN AND
POLYPEPTIDE SEQUENCE ANALYSIS
RELATED APPLICATION
This application claims priority on Provisional Application Serial No. 60/098,401, filed on August 31 , 1998. The contents of Provisional Application Serial No. 60/098,401, are incoφorated herein by reference.
FIELD OF THE INVENTION
The invention relates to apparatus and methods for protein or polypeptide sequencing. In particular, the invention relates to devices using continuous flow chambers for protein or polypeptide sequencing, and to methods for protein or polypeptide sequencing which use flow through chambers.
BACKGROUND
Recently several genomes have been sequenced, thereby defining hundreds of genes encoding new proteins of unknown biological function. The initial investigation of the biological activity of these proteins often begins with the production, sequencing, and characterization of the expressed recombinant protein. Sequence .analysis of a recombinant protein can provide rapid confirmation of its identity and the location and quantitation of cleavage sites within the protein. One method of N-terminal residue sequence analysis was introduced in 1950 by Pehr Edman
(ORGANIC CHEMISTRY, P 1129, Robert Thornton Morrison & Robert Neilson Boyd eds., 4th ed. 1983) (hereinafter "Edman Degradation Method"). The Edman Degradation Method is based on the reaction between an amino group and phenyl isothiocyante to form a substituted thiourea. Mild hydrolysis with acid selectively removes the N-terminal residue of a polypeptide as the phenylthiohydantoin. Thus, the degradation is a cyclic procedure, by which an amino acid residue is cleaved one at a time from the N-terminus of the polypeptide and identified as the phenylthiohydantoin derivative.
There are three steps in each cycle. The first step is coupling of phenylisothiocyanate (PITC) with the amino-terminal residue. The second step is cleavage of the amino-terminal residue via cyclization in acidic medium. The third step is conversion of the thiazolinone (ATZ) derivative formed during cleavage to generate the more stable thlohydantoin (PTH) derivative, which may be identified chromatographically (Burdon, R.H., ed., "Sequencing of Proteins and Peptides," in Laboratory Techniques in Biochemistry and Molecular Biology, vol. 9, chpt. 6, pp. 221-230 (1993)). Automated sequencing has greatly enhanced the speed with which proteins and polypeptides may be identified. Automated liquid phase protein and polypeptide sequencing began in 1967 with the introduction of the liquid phase spinning cup sequenator in which the tagging and cleavage reactions occur in a thin liquid film formed on the inside wall of a spinning reaction vessel. (Edman, P. and Begg, G., European J. of Biochem. 1:80-91 (1967). However, sample loss, particularly for short polypeptides, was a problem with this method.
Alternatively, the protein or polypeptide of interest may be trapped within a solid matrix or immobilized on a solid support matrix, such as a glass fiber matrix, a macoporous polystyrene matrix, or glass beads, by covalent attachment or by adsorption. The reagents and solvents are passed through a column packed with the solid matrix to which the polypeptide is attached.
In another method, the protein or polypeptide is blotted onto a polyvinylidene difluoride (PDVF) membrane following 2-D gel electrophoresis and the portion of the membrane to which the protein or polypeptide is attached is analyzed by the solid phase Edman Degradation Method.
To accurately identify a protein using the Edman Degradation Method, ten to twelve amino acid residues must be sequenced. Commercial sequencers, such as the model G1005A (HEWLETT
PACKARD™, Palo Alto, California) require 36-46 minutes to remove a single residue from the N- terminus of a protein. Consequently, the identification of each protein requires approximately 6 - 10 hours, thereby limiting the number of proteins that may be identified by a commercial sequencer each day. Totty et al. describes an Edman cycle requiring 25 minutes per residue obtained by raising reaction temperatures, reducing the size of the standard glass fiber matrix reaction cartridge, .and increasing the chromatographic flow rate (Totty, N.F. et al. Protein Sequence J_: 1215-1224 (1992)).
However, repetitive yield was reduced due to a decrease in carrier loading in keeping with the reduction in the glass fiber filter area and sample loadings (Totty, N.F. et al. (1992) supra). Further, increased chromatographic flow rate reduces detection sensitivity and column life.
An alternate method was described by Gooley et al. (Gooley, A.A. et al., 30 Electrophoresis 18:1068-1072 (1997)). To avoid the extended time necessary for sequencing 10 amino acid residues, Gooley et al. sequenced only 3 - 5 residues of each sample. Because protein identification cannot be done with so little sequence data, an amino acid analysis was also performed on each sample. The combination of on-line sequencing analysis with off-line amino acid analysis was required to confidently identify sample proteins. It was also proposed in Gooley et al. that other off-line techniques such as polypeptide masses, apparent protein mass (predicted by SDS-PAGE) and protein pi may be combined with 3 - 5 residue N-terminal sequence information to identify proteins. However, such mixed on-line and off-line analyses are cumbersome, time-consuming, and ultimately, more costly for the user.
Accordingly, there is a need for a device and method for rapidly and conveniently sequencing proteins and polypeptides. Additionally, there is a need for an automated and highly accurate device for sequencing proteins or polypeptides of unknown structure and biological function. Moreover, there is a need for a device for sequencing proteins and polypeptides which is relatively inexpensive to manufacture and utilize.
SUMMARY
An apparatus for rapidly and accurately sequencing samples of protein or polypeptides is provided herein. The apparatus includes a cartridge holder, a plurality of apparatus sample cartridges and a fluid connector. Each apparatus sample cartridge defines a chamber which receives one of the samples. Each apparatus sample cartridge is formed with a cartridge inlet and a cartridge outlet so that fluid can flow through the apparatus sample cartridge. As provided herein, the fluid connector selectively and independently connects a reagent port of a reagent delivery device in fluid communication with the cartridge inlet of each apparatus sample cartridge. This allows a single reagent delivery device to supply chemicals, i.e., reagents or solvents, to a plurality of apparatus sample cartridges. This reduces the cost to manufacture and operate the apparatus.
The fluid connector can be a multi-port radial valve having a connector inlet port, a connector outlet port, a plurality of supply ports and a plurality of return ports. The connector inlet port is attached in fluid communication with the reagent port of the reagent delivery device. Each apparatus sample cartridge is connected to one of the supply ports. Specifically, the cartridge inlet of each apparatus sample cartridge is connected in fluid communication with one of the supply ports of the valve. With this configuration, an independent path of fluid communication is selectively established from the reagent port of the reagent delivery device, through the connector inlet port and one of the supply ports and into the cartridge inlet of one of the apparatus sample cartridges.
The fluid connector also provides a path of fluid communication from the cartridge outlet of each apparatus sample cartridge to a conversion chamber and then to an analyzer. This allows a residue created by the interaction between the reagent and the sample to be transferred to the analyzer for analysis. Specifically, the cartridge outlet of each apparatus sample cartridge is connected in fluid communication with one of the return ports of the valve. The connector outlet port of the valve is connected in fluid communication with the conversion chamber and an analyzer port. In this manner, an independent path of fluid communication can be established from the cartridge outlet, through one of the return ports, through the valve outlet port and into the analyzer port. This allows a single analyzer to separately analyze the residue from multiple apparatus sample cartridges.
The cartridge holder retains the plurality of apparatus sample cartridges. As provided herein, the cartridge holder can be a printed circuit board having a plurality of holding stations. Each holding station is formed with a channel which is sized and shaped to receive one of the apparatus sample cartridges oriented substantially horizontal. Each holding station can also include a heater. The heater allows for individual heating of each individual apparatus sample cartridge to a desired temperature, from about ambient temperature to about 90 °C, inclusive.
As provided herein, the apparatus can be arranged as a module which is adapted for use with an existing sequencer having a plurality of sequencer sample cartridges. More specifically, the module includes a module base which retains the cartridge holder, the plurality of apparatus sample cartridges and the fluid connector as described above. Preferably, the module would include at least two and more specifically, at least five apparatus sample cartridges. In this embodiment, the module is connected to the sequencer in the place of one of the sequencer sample cartridges. Thus, the module expands the number of sample cartridges accessed by the sequencer and increases the throughput of the sequencer. Additionally, as provided herein, more than one module can be added to the sequencer to further expand the throughput of the sequencer.
In an alternative embodiment of the present invention, instead of using a valve, the sample cartridges are moved relative to a reagent outtake and an analyzer intake. In this embodiment, each holding station includes an inlet tube which is in fluid communication with one of the cartridge inlets and an outlet tube which is in fluid communication with one of the cartridge outlets. Further, the reagent outtake and the analyzer intake are secured to a port holder.
The reagent outtake is in fluid communication with the reagent port and the reagent delivery device while the analyzer intake is in fluid communication with the analyzer port and the analyzer. In use, the cartridge holder is moved to selectively align the inlet tube and the outlet tube of one holding station with the reagent outtake and the analyzer intake, respectively. With this configuration, reagent can be individually delivered to each sample cartridge from a single reagent delivery device by selectively moving the cartridge holder. Further, the residue from the sample cartridges can be individually transferred to the analyzer for analysis.
In operation, a sample is placed in the chamber of one of the apparatus sample cartridges. One or more reagents are selectively delivered to each apparatus sample cartridge. Residue from each apparatus sample cartridge is independently delivered to the analyzer. The operation of the apparatus provided herein is preferably done according to a rapid, approximately twenty minute (20 min) Edman cycle as provided below. In this manner, a plurality of samples are rapidly and accurately sequenced by the apparatus of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of this invention, as well as the invention itself, both as to its structure and its operation, will be best understood from the accompanying drawings, taken in conjunction with the accompanying description, in which similar reference characters refer to similar parts, and in which:
Figure 1 A is a top plan illustration of an apparatus having features of the present invention; Figure IB is a top plan illustration of an apparatus and a sequencer having features of the present invention;
Figure 1C is a top plan illustration of a plurality of apparatuses and a sequencer having features of the present invention; Figure 2 A is a perspective view of a holding station and a sample cartridge having features of the present invention;
Figure 2B is a cross-sectional view of a sample cartridge, a sample, a portion of a supply line and a portion of a return line having features of the present invention;
Figure 3 is a perspective view of an alternate embodiment of an apparatus having features of the present invention;
Figure 4 is a cross-sectional view taken on line 4-4 in Fig. 3;
Figures 5A-5L are chromatographic traces of PTH-amino acids following a sequence analysis pursuant to the present invention of mouse insulin-like growth factor binding protein 4 electroblotted onto PVDF; Figures 6A-6E are chromatographic traces of the PTH-amino acid standards separations using different chromatograph conditions in conjunction with the apparatus of the present invention;
Figures 7A and 7B are histograms illustrating an average amino acid yield of three analyses of PVDF electroblotted myoglobin (Fig. 7A) and beta-lactoglobulin (Fig. 7B) using the 20 minute Edman cycle provided herein, the single letter designations along the x-axis represent the amino acids detected; Figures 8A-8K are chromatographic traces of PTH-amino acids following sequence analysis of a 10 pmol sample of myoglobin electroblotted onto PVDF using a rapid Edman cycle described herein applied to 10 consecutive residues of the protein, the peak labeled " W" is an oxidized derivative of tryptophan; and
Figure 9 is a bar graph which highlights the repetitive yield accomplished using the apparatus and methods of the present invention on three separate protein samples.
DESCRIPTION
Before the present apparatus 10 and method are described, it is to be understood that this invention is not limited to the particular apparatus or processes described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting because the scope of the present invention is limited only by the appended claims.
Figure 1A illustrates a first embodiment of an apparatus 10, for rapid protein sequence analysis of multiple samples 12 having features of the present invention. The apparatus 10 illustrated in Figure
1A includes a reagent delivery device 14, a fluid connector 16, a conversion chamber or conversion chamber 17, an analyzer 18, a plurality of apparatus sample cartridges 20, a cartridge holder 22, and an apparatus controller 24. The apparatus 10 is useful for selectively and individually providing a plurality of chemicals, i.e., solvents or reagents 30 from a single reagent delivery device 14 to the plurality of sample cartridges 20. Subsequently, a residue 32 from each apparatus sample cartridge 20 is individually transferred to the conversion chamber 17 and then to the analyzer 18 for analysis. The apparatus 10 provided herein is particularly useful for the identification of an amino acid residue 32 released from the sample 12 following the coupling, cyclization, and conversion reactions in the individual, apparatus sample cartridges 20. Further, the apparatus 10 conveniently sequences a plurality of samples 12 using a single reagent delivery device 14. Additionally, the apparatus 10 allows for automated sample 12 sequencing which is very accurate and relatively rapid. Each sample 12 is typically a protein or a polypeptide of unknown structure and biological function. Each protein or polypeptide can be blotted onto a polyvinylidene fluoride (PVDF) membrane following 2-D gel electrophoresis. Subsequently, a portion of the membrane containing the sample 12 can be placed within one of the apparatus sample cartridges 20. Each sample 12 can be absorbed or covalently attached to the membrane so that the protein or polypeptide is not dislodged during the coupling and cyclization reactions. The protein or polypeptide of interest may have been isolated from other proteins or polypeptides by gel electrophoresis and a portion of the gel containing the protein or polypeptide is cut from the gel and transferred to the membrane.
The reagent delivery device 14 delivers a predetermined volume of one or more selected solvents and reagents 30 to the apparatus sample cartridges 20. Examples of suitable reagent delivery devices 14 include, but are not limited to, HEWLETT-PACKARD Model 61005™ and PROCISE 494™ and PROCISE 473™ sold by Applied Biosystems of PE Biosystems located in Foster City, California. The reagent delivery device 14 illustrated in Figure 1A includes a single reagent port 36 which delivers the solvents and reagents 30. Suitable reagents and solvents 30 are outlined in the examples provided below. The conversion chamber 17 initially receives the residue 32 from each sample cartridge 20.
The conversion chamber 17 is also referred to as a conversion flask. The conversion chamber 17 is preferably a container which is lined or made of a chemically inert material such as polytetraflouroethylene or glass. A suitable polytetraflouroethylene is sold under the trademark Teflon™. The conversion chamber 17 preferably includes a heater (not shown) which heats the conversion chamber 17 to between approximately ambient to 70°C, inclusive. In the conversion chamber 17, reagents 30 and residues 32 in which the thiazolinone (ATZ) derivative formed during cleavage of the amino terminal residue is converted to the detectable more stable thiohydantoin (PTH) derivative. Preferably, the conversion chamber 17 is downstream of the sample cartridge 10 and upstream of the analyzer 18. The analyzer 18 includes an analyzer port 38 for receiving the residue 32 from the conversion chamber 17. The analyzer 18 can include, but is not limited to, a gas or high pressure liquid chromatographic column 40, and/or an amino acid derivative detection system 42 (such as a light, mass, charge or other physical detection technique available at the time of analysis). The amino acid derivative detection system 42 can preferably detect an amino acid derivatized and released from the sample 12 of interest with sufficient sensitivity to reproducibly identify a given amino acid above the background signal. The fluid connector 16 individually and independently connects each of the apparatus sample cartridges 20 to the reagent port 36 and the analyzer port 38 via the conversion chamber 17. In the embodiment illustrated in Figure 1A, the fluid connector 16 is a multi-port, multi-position, radial valve 43 which includes a connector inlet port 44 which is in fluid communication with the reagent port 36. More specifically, in the embodiment illustrated in Figure 1A, a reagent tube 46 connects the reagent port 36 in fluid communication with connector inlet port 44.
The valve 43 illustrated in Figure 1A also includes six supply ports 48. The supply ports 48 provide individual and independent paths for solvents or reagents 30 to flow from the connector inlet port 44 to each of the apparatus sample cartridges 20. A plurality of supply tubes 50 are used so that each supply port 48 is in fluid communication with one of the sample cartridges 20. More specifically, a separate supply tube 50 is used to connect one of the supply ports 48 to one of the apparatus sample cartridges 20. Stated another way, one of the supply tubes 50 extends between one of the supply ports 48 and a cartridge inlet 52 to one of the apparatus sample cartridges 20. This allows solvents and reagents 30 from the reagent delivery device 44 to be independently delivered to each individual apparatus sample cartridge 20. The valve 43 also includes a connector outlet port 54 in fluid communication with the analyzer port 38 via the conversion chamber 17. More specifically, in the embodiment illustrated in Figure 1A, an analyzer tube 56 connects the analyzer port 38 in fluid communication with connector outlet port 54 via the conversion chamber 17. The valve 43 illustrated in Figure 1A, also includes six return ports 58. The return ports 58 provide individual and independent paths for residue 32 to flow from each apparatus sample cartridge 20 to the connector outlet port 54. A plurality of return tubes 60 are used so that each return port 58 is in fluid communication with one of the apparatus sample cartridges 20. More specifically, a separate return tube 60 is used to connect a cartridge outlet 62 of one of the sample cartridges 20 to one of the return ports 58. Stated another way, one of the return tubes 60 extends between one of the cartridge outlets 62 and one of the return ports 58. This allows the residues 32 from each sample cartridges 20 to be delivered independently to the conversion chamber 17 and then the analyzer 18.
The valve 43 is selectively controlled to establish an individual and independent path of fluid communication from the reagent delivery device 14, through the connector inlet port 44, through one of the supply ports 48, and to one of the sample cartridges 20. Somewhat similarly, the valve 43 is also selectively controlled to establish an individual and independent path of fluid communication from one of the apparatus sample cartridges 20, through one of the return ports 58, through the connector outlet port 54, to the conversion chamber 17 and to the analyzer 18. Preferably, the supply ports 48 and the return ports 58 of the valve 43 are equally spaced apart and oriented radially around the body of the valve 43. With this design, the fluid path from the connector inlet port 44 to each of the supply ports 48 is approximately the same length. Similarly, the fluid path from each return port 58 to the connector outlet port 54 is approximately the same length. This improves the repeatability of the apparatus 10. A suitable valve 43 for the embodiment illustrated in Figure 1 A is a 14-port stainless steel automated valve, sold by Valco Instruments Co., Inc., located in Houston, Texas under the model number EMT-CST-UWTF.
The reagent tube 46, the supply tubes 50, the return tubes 60, and the analyzer tube 56 are preferably made of a chemically inert material such as polytetrafluoroethylene. A suitable polytetraflouroethylene material is sold under the trademark Teflon™. Suitable tubing has an inner diameter of between approximately 0.3mm and 0.5 mm and an outer diameter of approximately 1/16 inch.
The apparatus sample cartridges 20 retain the plurality of samples 12 during sequencing. Each apparatus sample cartridge 20 includes a chamber 64 which is sized and shaped to receive one of the samples 12. Each apparatus sample cartridge 20 also includes the cartridge inlet 52 and the cartridge outlet 62 to allow for fluid flow through the chamber 64.
Preferably, each cartridge inlet 52 is connected directly to one of the supply tubes 50 and each cartridge outlet 62 is connected directly to one of the return tubes 60 for quick and easy attachment and detachment of the apparatus sample cartridges 20. Additionally, the attachments between (i) the supply tube 50 and the apparatus sample cartridge 20 and (ii) the return tube 60 and the apparatus sample cartridge 20 preferably form a liquid-tight and gas-tight seal so that the sample 12, the reagents 30 and residue 32 are not appreciably lost from the apparatus sample cartridge 20. Preferably, each apparatus sample cartridge 20 is also quickly and easily inserted into or removed from the cartridge holder 22 to facilitate sample 12 changes. The apparatus samples cartridges 20 are preferably made of a chemically inert material, such as glass or polytetraflouroethylene, sold under the trademark Teflon™.
Referring to Figures 2 A and 2B, each apparatus sample cartridge 20 can be a hollow tube which receives the membrane and sample 12. Further, each apparatus sample cartridge 20 can have an outer diameter of approximately 1/8 inch and an inner diameter of approximately 1/16 inch. As illustrated in Figure 2B, this sizing allows the 1/16 inch outer diameter supply tube 50 and return tube 60 to fit tightly within the apparatus sample cartridge 20 and form a liquid-tight and gas-tight seal. Further, this arrangement allows the supply tube 60 and the return tube 50 to retain the sample 12 within the apparatus sample cartridge 20. Each apparatus sample cartridge 20 can be between approximately 15- 20 mm long.
The cartridge holder 22 retains the plurality of apparatus sample cartridges 20 spaced apart. The cartridge holder 22 illustrated in Figure 1A includes a holder body 66 and a plurality of spaced apart holding stations 68. In the embodiment illustrated in Figure 1A, the holder body 66 is a flat, planer shaped circuit board. Alternate embodiments of the holder body 66 can be utilized. Each holding station 68 is adapted to retain one of the apparatus sample cartridges 20.
Referring to Figure 2 A, each holding station 68 can include a generally rectangular shaped holding block 70, formed with a channel 72 which is sized and shaped to retain one of the apparatus sample cartridges 20 press fit into the channel 72. Alternately, those skilled in the art will recognize other ways to secure each apparatus sample cartridge 20 to one of the holding blocks 70.
Preferrably, each holding station 68 includes a heater 74 for individually heating each apparatus sample cartridge 20 to expedite the reactions within the sample cartridges 20. The heater 74 transfers heat to each holding station 68 surrounding the apparatus sample cartridge 20. The heater 74 preferably heats the sample 12 to a range of from approximately ambient temperature to approximately 90°C, inclusive. A suitable heater 74 can be a thermofoil strip attached in thermal communication to each holding block 70. A suitable thermofoil strip is the model HK913L sold by Minco, located in Minneapolis, Minnesota. In a preferred embodiment, each holding block 70 is made of aluminum. The aluminum provides a heat sink to maintain uniform temperature around the sample cartridge 20.
Each heater 74 of each holding block 70 is preferably designed to be individually activated and individually controlled by current from a source of electrical current 76. In the embodiment illustrated in Figure 1A, each heater 74 (shown in Figure 2 A) is individually in electrical communication with the source of electrical current 76 with a separate electrical line (not shown). The separate electrical lines are positioned side-by-side and form a bundle of electrical lines 78 illustrated in Figure 1A. Thus, current may flow from the source of electrical current 76 through each electrical line to each individual heater 74 so that each heater 74 increases the temperature of the apparatus sample cartridge 20 to a desired temperature.
Further, each holding station 68 can include an independent thermocouple sensor 75 (shown in Figure 2A) which measures the temperature of the holding station 68 and/or the apparatus sample cartridge 20. Information from each individual thermocouple sensor 75 can independently transferred to the apparatus controller 24 (shown in Figure 1A). With information regarding the temperature, the apparatus controller 24 can control the source of electrical current 76 so that each heater 74 heats the apparatus sample cartridge 20 to the desired temperature and at a desired time in the reaction cycle. Each heater 74 is preferably individually controlled by the apparatus controller 24. As provided herein, the apparatus controller 24 can control each heater 74 so that the apparatus sample cartridges 20 are all at the same or different temperatures.
The apparatus sample cartridges 20 are preferrably maintained in a horizontal orientation 80 by the cartridge holder 22 to minimize reagent evaporation and to allow maximum contact between reagents 30 and sample 12. Stated another way, the apparatus sample cartridges 20 are positioned so that a longitudinal axis 81 of each apparatus sample cartridge 20 is substantially horizontal. The horizontal orientation 80 of each apparatus sample cartridge 20 provides superior sequence analysis results because of improved contact between the sample 12 and the reagents and solvents 30. The horizontal orientation 80 of each apparatus sample cartridge 20 also enhances repetitive yield and improves sequence analysis accuracy.
Referring back to Figure 1A, the apparatus controller 24 is electronically connected to the fluid connector 16, i.e., the valve 43 with electrical line 82. The controller 24 controls the position of the valve 43 so that (i) the solvents or reagents 30 are independently delivered to the appropriate apparatus sample cartridge 20, and/or (ii) residue 32 from the appropriate apparatus sample cartridge 20 is independently transferred to the conversion chamber 17 and then the analyzer 18. As provided herein, the apparatus controller 24 can control a pneumatic and/or electrical motor 84 which moves the valve 43. Basically, the apparatus controller 24 controls the motor 84 so that the appropriate supply port 48 is independently in fluid communication with the connector inlet port 44 or the appropriate return port 58 is independently in fluid communication with the conversion chamber 17. Thus, the fluid controller 24 controls the flow to and from each apparatus sample cartridge 20. Additionally, as provided above, the apparatus controller 24 is electrically connected to each holding station 68 so that the apparatus controller 24 can individually control each heater 74. Further, the apparatus controller 24 can include a display 85 which displays which sample 12 is being sequenced. In particular, the display 85 in Figure 1A includes a plurality of lights which illuminate to identify which sample 12 is being sequenced.
Additionally, the apparatus 10 can include a plurality of optical sensors 86 for monitoring flow through the supply tubes 50 or the return tubes 60 to determine and/or detect whether there is fluid flow through each apparatus sample cartridges 20. In Figure 1A, each optical sensor 86 monitors flow through one of the return tubes 60, near one of the apparatus sample cartridges 20. With this arrangement, each optical sensor 86 can monitor or detect when the chemicals 30 are positioned in one of the apparatus sample cartridges 20. The optical sensors 86 illustrated in Figure 1A are electrically connected to a jack connection 88, which are electrically connected to the apparatus controller 24. Information from the optical sensors 86 can subsequently be individually transferred to the apparatus controller 24. This allows the apparatus controller 24 to monitor flow through each apparatus sample cartridge 20.
Importantly, the embodiment illustrated in Figure 1A includes six apparatus sample cartridges 20 which sequence six separate samples 12. It is to be appreciated, however, that the apparatus 10 of Figure 1A can be designed to include more than six or less than six sample cartridges 20 provided the fluid connector 16 has a supply port 48 and return port 58 for each apparatus sample cartridge 20 or a second fluid connector (not shown) is utilized. Further, the cartridge holder 22 can include more than or less than six holding station 68.
Figure IB illustrates that the apparatus 10 can be arranged as a module 200 and used in conjunction with a commercial sequencer 202. In particular, the module 200 illustrated in Figure IB includes a module base 204 which retains the various components of the apparatus 10. More specifically, the fluid connector 16, the apparatus sample cartridges 20, the cartridge holder 22, and the apparatus controller 24 are all secured to the module base 204.
In the embodiment illustrated in Figure IB, the apparatus sample cartridges 20, the cartridge holder 22, the apparatus controller 24, and the fluid connector 16 are similar to that described above in the discussion of Figure 1A. However, the apparatus 10 is arranged in this embodiment as a module 200 which can be added to the commercial sequencer 202. Although six apparatus sample cartridges 20 are illustrated in Figure IB, the number of apparatus sample cartridges 20 for the module 200 can be more than six or less than six.
A simplified illustration of a commercial sequencer 202 and module 200 is provided in Figure IB. It should be noted that the sequencer 202 typically includes numerous additional components which are not illustrated in Figure IB and that the sequencer 202 is typically much larger than the module 200. The sequencer 202 illustrated in Figure IB includes (i) a sequencer base 208, (ii) a sequencer reagent delivery device 210, (iii) a horizontally oriented, reagent port valve 212, (iv) four, sequencer sample cartridges 214, (v) a horizontally oriented analyzer port valve 216, (vi) a sequencer conversion chamber 218, (vii) a sequencer analyzer 220, and (viii) a sequencer controller 222. The sequencer reagent delivery device 210 can be similar to the reagent delivery device 14 described above. Similarly, the sequencer conversion chamber 218 and the sequencer analyzer 220, respectively can be similar to the conversion chamber 17 and analyzer 18 described above.
Uniquely, in this embodiment, one of the sequencer sample cartridges 214 is disconnected from the sequencer 202. Instead, the fluid connector 16 of the module 200 is connected in its place. Specifically, one of the reagent outlet ports 224 is connected (with line designated 46) in fluid communication with the connector inlet port 44 of the fluid connector 16 of the module 200. Somewhat similarly, the connector outlet port 54 of the module 200 is connected (with line 56) in fluid communication with one of the analyzer inlet ports 226 of the sequencer 202. Further, the apparatus controller 24 is electrically connected, via line 228 to the sequencer controller 222. This allows the sequencer controller 222 to control the fluid connector 16.
With this configuration, the module 200 is used to expand the throughput of the sequencer
202. More specifically, the sequencer 202 illustrated initially utilized only four sequencer sample cartridges 214. Now, with the addition of the module 200, having six apparatus sample cartridges 20, a total of nine samples 12 can be sequenced by the sequencer 202. Thus, the module 200 can be used to increase the throughput of the sequencer 202.
Figure 1C illustrates another embodiment of the present invention. In particular, in Figure 1C, the commercial sequencer 202 is connected to four, separate individual modules 200. Each module 200 is represented as a rectangle in Figure lC. However, each module 200 illustrated in Figure 1C can be substantially similar to and include all of the components of the module 200 illustrated in Figure IB and described above. In this embodiment, (i) each reagent outlet port 224 of the sequencer reagent delivery device 210 is connected to one of the modules 200, instead of the sequencer sample cartridges 214, (ii) each analyzer inlet port 226 is connected to one of the modules 200 instead of the sequencer sample cartridges 214, and (iii) each module 200 is electrically connected to the sequencer controller 222. With this set up, depending upon the design of the module 200, throughput of the sequencer 202 can be greatly increased. More specifically, if each module 200 includes six apparatus sample cartridges 20, the throughput of the sequencer 202 and four modules 200 would be adapted to process twenty-four samples 12. Further expansion is readily possible by increasing the number of apparatus sample cartridges 20 in each module 200.
Figure 3 illustrates another alternative embodiment of an apparatus 10 having features of the present invention. In this embodiment, the cartridge holder 20 also includes a plurality of spaced apart holding stations 68 for holding a plurality of spaced apart, apparatus sample cartridges 20. However, in this embodiment, a valve 43 (not shown in Figure 3) is not used to direct the particular reagents 30 from the reagent port 36 to each individual apparatus sample cartridge 20, and the residue 32 from each apparatus sample cartridge 20 to the conversion chamber 17. Instead, referring to Figure 4, a reagent outtake 87 and an analyzer intake 89 are secured to a port holder 90. The reagent outtake 87 is in fluid communication with the reagent delivery device 14 and the analyzer intake 89 is in fluid communication with the analyzer 18 via the conversion chamber 17. In this embodiment, one of the holders, i.e. the port holder 90 or the cartridge holder 22 is moved relative to the other holder so that the reagent outtake 87 and the analyzer intake 89 are selectively connected in fluid communication with one of the apparatus sample cartridges 20. The cartridge holder 22 illustrated in Figure 3 is shaped similar to a carousel and includes twelve, spaced apart, holding stations 68. Further, in this embodiment, the cartridge holder 22 is selectively rotated about a central axis 92 by a mover 94 (shown in phantom in Figure 3). The mover 94 is mounted to the cartridge holder 22 below a bottom side 96 of the cartridge holder 22. With this configuration, the cartridge holder 22 and apparatus sample cartridges 20 can be selectively moved by the mover 94 relative to the port holder 90. The mover 94 can be a pneumatic and/or electrical motor or other means for moving the cartridge holder 22 relative to the port holder 90. In the embodiment illustrated in Figure 3, the mover 94 rotates the cartridge holder 22 to twelve alternate positions so that the twelve apparatus sample cartridges 20 are individually brought into fluid communication with the reagent outtake 87 and the analyzer intake 89. It is understood that linear movement of the cartridge holder 22 is capable of accomplishing the same result as the rotation of the cartridge holder 22 described herein, and is considered an embodiment of the invention. Moreover, the cartridge holder 22 can be designed to retain more than twelve or less than twelve apparatus sample cartridges 20. Further, the apparatus 10 could be designed so that the reagent outtake 87 and the analyzer intake 89 are moved relative to the cartridge holder 22 and the sample cartridges 20.
In the embodiment illustrated in Figures 3 and 4, each holding station 68 includes a separate inlet tube 98 connected to the cartridge inlet 52 of the apparatus sample cartridge 20 and separate outlet tube 100 connected to the cartridge outlet 62 of the apparatus sample cartridge 20. Each inlet tube 98 inserts through an inlet hole 102 in the cartridge holder 22 while each outlet tube 100 inserts through an outlet hole 104 in the cartridge holder 22. Each inlet tube 98 includes an inlet tube distal end 106 which is secured to the cartridge holder 22 substantially flush with the bottom side 96 of the cartridge holder 22. Similarly, each outlet tube 100 includes an outlet tube distal end 108 which is secured to the cartridge holder 22 substantially flush with the bottom side 96 of the cartridge holder 22. The distal ends 106, 108 are spaced apart a fixed end distance 110. Each inlet tube 98 and outlet tube 100 are preferably made of a chemically inert material, such as Teflon™. For the embodiments provided herein, each inlet tube 98 and outlet tube 100 has an inner diameter of between approximately 0.3mm and 0.5 mm and an outer diameter of approximately 1/16 inch. However, those skilled in the art will recognize that alternate sizes can be utilized.
Preferably, an inlet tube seal I l ia encircles the inlet tube distal end 106 and extends away from the bottom side 96 of the cartridge holder 22. Similarly, an outlet tube seal 111b encircles the outlet tube distal end 108 and extends away from the bottom side 96 of the cartridge holder 22. Additionally, each holding station 68 can include a heater 74 and a thermocouple sensor 75 as described above. The embodiment illustrated in Figure 3 also provides a unique way to electrically connect each heater 74 and thermocouple sensor 75 to the source of electrical current 76 and the apparatus controller 24. In particular, each holding station 68 can include at least a pair of spaced apart heater electrical contacts 112a and a pair of spaced apart thermocouple electrical contacts 112b which are positioned on an outer perimeter 114 of the cartridge holder 22. Each electrical contact 112a is electrically connected with electrical wires 116a to one of the heaters 74. Each thermocouple electrical contact 112b is electrically connected with electrical wires 116b to one of the thermocouple sensors 75. In this embodiment, the source of electrical current 76 includes a resilient beam 1 18 which is urged against and contacts the outer perimeter 114 of the cartridge holder 22. The beam 118 includes at least four beam electrical contacts 120 which are spaced apart. Two of the beam electrical contacts 120 are adapted to contact the heater electrical contacts 112a of one of the heaters 34 and two of the beam electrical contacts 120 are adapted to contact the thermocouple electrical contacts 112b. The beam electrical contacts 120 are electrically connected to the source of electrical current 76 and the apparatus controller 24 with source wires 122. This allows the apparatus controller 24 to monitor the temperature of each heater 74 and adjust the current to each heater 74 to adjust the temperature of each heater 74. The beam 1 18 illustrated in Figure 3 includes vertical portion 124 and a transverse portion 126 which cantilevers away from the vertical portion 124 and contacts the outer perimeter 114 of the cartridge holder 22.
Figure 4 illustrates the interaction between the holding station 68, the reagent outtake 87 and the analyzer intake 89 in more detail. In particular, in this embodiment, the reagent outtake 87 and the analyzer intake 89 are mounted in a block shaped port holder 90. The reagent outtake 87 includes a reagent outtake distal end 128 which is mounted substantially flush with an upper surface 130 of the port holder 90. A reagent outtake seal 132 encircles the reagent outtake distal end 128 and extends away from the upper surface 130 of the port holder 90. Similarly, the analyzer intake 89 includes an analyzer outtake distal end 134 which is mounted substantially flush with the upper surface 130 of the port holder 90. An analyzer intake seal 136 encircles the analyzer intake distal end 134 and extends away from the upper surface 130 of the port holder 90. The reagent outtake distal end 128 and the analyzer intake distal end 134 are spaced apart substantially the same distance as the inlet tube distal end 106 and the outlet tube distal end 108.
The reagent outtake seal 132 interacts with the inlet tube seal 11 la to establish a fluid tight path of fluid communication between the reagent outtake 87 and the inlet tube 98. Similarly, the analyzer intake seal 136 interacts with the outlet tube seal 111b to provide a fluid tight path of fluid communication between the analyzer intake 89 and the outlet tube 100. The inlet tube seal I l ia, the outlet tube seal 111b, the reagent outtake seal 132 and the analyzer intake seal 136 can each be an "O" ring type seal.
With the embodiment provided in Figure 3, the cartridge holder 22 is moved by the mover 94 so that each sample cartridge 20 is individually aligned with the reagent outtake 87 and the analyzer intake 89, the electrical contacts 112a, 112b are aligned with the source electrical contacts 120.
EXAMPLES
The following examples are set forth so as to provide those of ordinary skill in the art with additional disclosure of the apparatus 10 of the invention and how to use it. The disclosure is not intended to limit the scope of what the inventor regards as his invention. Efforts have been made to insure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees centigrade, and pressure is at or near atmospheric. Example 1 : Automated Sequence Analysis Device. The chromatograms obtained from mouse insulin-like growth factor binding protein 4 (IGFBP-4), using the apparatus 10 of the invention are illustrated in Figs. 5A-5L. The partial N-terminal sequence of the protein was verified by the rapid Edman cycle method described herein to be DEAIHCPPCSE (SEQ ID NO:l) (Schuller A.G. et al. (1994) Mol. Cell. Endocrinol. 104:57-66, GenBank accession number 148600). The device according to the present invention was used with rapid Edman cycles to sequence consecutive residues of the protein.
Example 2: Methods of Analysis Using Rapid Edman Cylces.
An Edman degradation cycle for protein sequencing typically involves coupling PITC with the amino terminal residue; cleavage of the amino terminal residue via cyclization in acidic medium; and conversion of the resultant ATZ derivative to a more stable PTH derivative that may be detected and identified. The time required for one cycle of Edman degradation is dependent upon the time required to complete both the Edman chemistry and the PTH analysis. The PTH chromatographic separation has been a primary limiting factor of cycle speed. Others have approached this problem by using higher flow rates at the expense of lower sensitivity (Totty et al. (1992) supra).
Chromatographic Figues 6A and 6B compare separations using a short column (0.2 cm x 15 cm, Fig. 6A) and a conventional chromatographic column (0..2 cm x 22 cm, Fig. 6B), each packed with 5 μm C18 resin. The standard single letter designation for the amino acids are used to indicate the identity of the PTH-amino acid peaks. According to a method disclosed herein, increased speed, as well as increased sensitivity, are achieved through the use of a short chromatographic column (i.e., a 15 cm column instead of the standard 22 cm column). Figs. 6C, 6D and 6E respectively compare separations using a 15 cm, 12.5 cm and 10 cm chromatographic column (each 0.2 cm diameter), respectively, packed with 3 μm C18 resin. The time required to equilibrate the chromatographic column was also reduced by using a higher flow rate of 375 μl/min, rather than the standard flow rate of 325 μl/min of commercial devices, such as the PROCISE 140C™, sold by Perkin Elmer- Applied Biosystems Division. Detection sensitivity increased because the short chromatographic column limited peak spreading.
To equilibrate the chromatographic column for separation and identification of a derivatized amino acid residue, the flow rate was programmed to be a specified rate at the beginning of each reaction cycle (which cycle includes coupling, cyclization and conversion of the N-terminal amino acid of the protein or polypeptide of interest). The resultant flow gradient that occurs during the first minute of the separation as the flow rate rapidly decreases to the separation flow rate of 325 μl/min had no affect on retention time reproducibility of the derivatized amino acids. The 15 cm chromatographic columns provided separations for an average of 2,000 to 3,000 injections before peak resolution began to decrease. Table 1 A lists chromatographic separation gradient conditions useful in the invention.
TABLE 1A Mobile Phase Gradient for Chromatographic Separation
Coupled to a Rapid Edman Cycle
Figure imgf000017_0001
Solvent A of the gradient was 3.5% tetrahydrofuran (THF) in aqueous buffer comprising 2%
Pre-Mix™ buffer (Perkin-Elmer/Applied Biosystems). Solvent B was acetonifrile. Optionally solvent
B may be 11-12.5% isopropanol in acetonifrile, where isopropanol may be useful to enhance separation of the PTH-tryptophan derivative from its oxidized form. The target pressure is approximately 1500 psi and data collection time was approximately 20 min.
Further improvement in rapid Edman cycle protein sequence analysis was achieved by shortening the time for chromatographic analysis (Henzel, W.J. et al., Analytical Biochemistry 267:148- 160 (1999)). In addition to the 15 cm chromatographic column described above, Figs. 6D and 6E show that 12.5 cm and 10 cm chromatographic columns packed with 5 μm or 3 μm C18 resin reduce analysis time while improving resolution and sensitivity. Separation of PTH-amino acid derivatives was also improved by modifications in chromatographic gradient, flow rate, and equilibration parameters. Separation of 1 picomole of PTH-amino acid standards was compared on 15 cm, 12.5 cm, and 10 cm chromatographic columns packed with Haisil HL™ 3 μm C18 PTH resin (Higgins Analytical, Inc) (See Table IB and Figs. 6C, 6D, and 6E). The highest detection sensitivity was obtained using a Haisil HL™ 3 μm C18 PTH resin in a 0.20cm x 10cm chromatographic column. Column equilibration time was reduced using a higher flow rate of 300 μl/min for 8 minutes, then lowering the flow rate to 275 μl/min at 0.2 minutes in the chromatographic gradient.
TABLE IB Mobile Phase Gradient for Chromatographic Separation on 15 cm, 12.5 cm and 10 cm columns
15 cm column 12.5 cm column 10 cm column
Figure imgf000018_0001
Solvent A of the gradient was 3.5% tetrahydrofuran (THF) in aqueous buffer comprising 2-4% Pre-Mix™ buffer concentrate (Perkin-Elmer/Applied Biosystems). To each liter of Solvent A was added 15-25 μl acetone, 0.5 ml acetic acid, and 0.1 ml 1M Na2P04. For the 10 cm column, THF was 5% to resolve PTH-Gln and PTH-Thr. Solvent B was acetonifrile/ 11-12.5% isopropanol. Column temperature was 55°C.
Alterations in Edman cycle parameters also contributed to achievement of a rapid 20-minute Edman cycle pursuant to the present invention. The duration of the Edman cycle was decreased by increasing the temperature of the coupling, cyclization and conversion reactions. The methods disclosed herein indicate appropriate solvents useful in the reactions and washes such that the more rapid reactions and separations remained reproducible and quantitative. Table 2 compares the reaction conditions of the current commercially available PROCISE™ cycle with a 20-minute rapid, Edman cycle pursuant to the present invention useful with the apparatus 10 of the invention.
TABLE 2
A Comparison of the Current PROCISE™ PVDF versus A Rapid Edman Cycle Method
Figure imgf000019_0001
Figs. 7A and 7B are histograms illustrating the average amino acid yield of three analysis of PVDF electroblotted myoglobin (Fig. 7A) and beta-lactoglobulin (Fig. 7B) using the 20 minute Edman cycle provided herein. The single letter designations along the x-axis represent the amino acids detected.
Figs. 8A-8K are chromatographic traces of PTH-amino acids following sequence analysis of a 10 pmol sample of myoglobin electroblotted onto PVDF using a rapid Edman cycle as provided herein applied to 10 consecutive residues of the protein. According to a preferred method for use with the apparatuses of the invention, each Edman cycle as provided herein requires less than approximately 20 minutes, while the commercial method requires 33 minutes. The temperature of the coupling and cleavage reactions of the present invention was increased from 48 °C to 55 °C, and the conversion temperature was increased to 75 °C. These cycles also incorporated an acetone wash (designated "X3" in Table 3) after the coupling reaction to reduce the diphenylthiourea (DPTU) by-product of the reactions involving aniline and ethyl acetate, which DPTU by-product co-elutes with the PTH derivative of glutamine. Reducing the amount of DPTU by-product allowed a significant decrease in cycle time because the cycles of conventional methods bracket deliveries of ethyl acetate with deliveries of butyl chloride, thereby preventing the accumulation of this aniline derivative, but resulting in longer cycle times. In addition, the conversion cycle of the invention utilizes acetonifrile (designated "X2" in Table 3) as the solvent to wash the conversion chamber 17. Acetonifrile is a more efficient solvent for removing by-products from the conversion flask than a conventionally-used aqueous acetonifrile solution (Totty, N.F. et al., supra). Fast Edman Cycle Method 1 :
The programmed steps of a 20-minute Edman cycle, Edman Method 1, are listed in Table 3. Method 1 refers to a cycle of steps that may be used with reagent delivery devices for delivering a limited number of reagents or solvents to the sample cartridge and conversion flask 17 for reaction and wash steps.
TABLE 3A
Method 1
Program Steps For A Rapid Edman Cycle
BEGIN CYCLE
Step Function Name Elapsed Time
1 Begin :00
2 Wash (Cartridge) Small loop SI :05
3 Flush Small Loop (Cartridge) : 15
4 Flush Cartridge Solvent Block :25
5 Flush Input Block :35
6 Dry Cartridge (top) 1:35
7 Flush Cartridge Solvent Block 1 :45
8 Wash Cartridge Solvent Block SI 1 :50
9 Flush Cartridge Solvent Block 2:00
10 Flush Cartridge Reagent Block 2:10
1 1 Wash (Cartridge) Small loop SI 2:15
12 Flush Small Loop (Cartridge) 2:25 Step Function Name Elapsed Time
13 Flush Input Block 2:35
14 Del S2, Carfridge (sensor) 2:50
15 Cartridge Wait 2:55
16 Del S2, Carfridge (top) 3:05
17 Cartridge Wait 3:10
18 Del S3, Cartridge (top) 3:20
19 Cartridge Wait 3:25
20 Del X3, Cartridge (top) 3:30
21 Cartridge Wait 3:35
22 Del S3, Carfridge (top) 3:40
23 Dry Cartridge (top) 4:40
24 Flush Cartridge Reagent Block 4:50
25 Flush Input Block 5:00
26 Del R2g, Cartridge (top) 5:30
27 Flush Small Loop (Cartridge) 5:40
28 Load RI, Cartridge (small loop) 5:52
29 Dry Cartridge (top) 6:12
30 Flush Small Loop (Cartridge) 6:22
31 Flush Cartridge Reagent Block 6:32
32 Del R2g, Cartridge (top) 8:32
33 Flush Small Loop (Cartridge) 8:42
34 Load RI, Cartridge (small loop) 8:54
35 Dry Cartridge (top) 9:14
36 Flush Small Loop (Cartridge) 9:24
37 Flush Cartridge Reagent Block 9:34
38 Del R2g, Cartridge (top) 11 :34 Step Function Name Elapsed Time
39 Wash (Cartridge) Small loop SI 1 1:39
40 Flush Small Loop (Cartridge) 11:49
41 Cartridge Reagent Block wash SI 11 :54
42 Flush Cartridge Reagent Block 12:04
43 Dry Cartridge (top) 13:04
44 Del S2, Cartridge (sensor) 13:19
45 Cartridge Wait 13:24
46 Del S2, Cartridge (top) 13:34
47 Cartridge Wait 13:39
48 Del S3, Cartridge (top) 13:49
49 Cartridge Wait 13:54
50 Del X3, Cartridge (top) 13:59
51 Cartridge Wait 14:04
52 Del S3, Cartridge (top) 14: 14
53 Dry Cartridge (top) 15:14
54 Flush Cartridge Reagent Block 15:24
55 Flush Input Block 15:34
56 End 15:34
RI 5% phenylisothiocyanate in hepane
R2 Methyl piperidine in n-propanol and water (25:60:15)
R3 Trifluoracetic acid (TFA)
R4 25% TFA in water
R5 PTH standard in acetonifrile (1 pmol/50 μl)
SI Heptane
S2 Ethyl acetate
S3 Butyl chloride
S4 7.5% acetonifrile in water
X2 Acetonifrile
X3 Acetone TABLE 3B
Method 1
Program Steps For A Rapid Edman Cycle
CARTRIDGE CYCLE
Step Function Name Elapsed Time
1 Begin :00
2 Flush Input Block :10
3 Flush Cartridge Reagent Block :20
4 Del R2g, Cartridge (top) :40
5 Flush Small Loop (Cartridge) :50
6 Load RI, Cartridge (small loop) 1 :02
7 Dry Carfridge (top) 1:22
8 Flush Small Loop (Cartridge) 1:32
9 Flush Cartridge Reagent Block 1:42
10 Del R2g, Carfridge (top) 3:42
11 Flush Small Loop (Carfridge) 3:52
12 Load RI, Cartridge (small loop) 4:04
13 Dry Carfridge (top) 4:34
14 Flush Small Loop (Cartridge) 4:44
15 Flush Cartridge Reagent Block 4:54
16 Del R2g, Carfridge (top) 6:54
17 Flush Cartridge Reagent Block 7:04
18 Dry Carfridge (top) 8:04
19 Cartridge Reagent Block Wash SI 8 :09
20 Flush Cartridge Reagent Block 8:19
21 Flush Input Block 8:29
22 Wash (Carfridge) Small loop SI 8:34
23 Flush Small Loop (Cartridge) 8:44 Step Function Name Elapsed Time
24 Del S2, Cartridge (sensor) 8:59
25 Cartridge Wait 9:04
26 Del S2, Cartridge (top) 9:09
27 Cartridge Wait 9:14
28 Del S3, Cartridge (top) 9:24
29 Cartridge Wait 9:29
30 Del X3, Cartridge (top) 9:34
31 Cartridge Wait 9:39
32 Del S3, Cartridge (top) 9:44
33 Cartridge Wait 9:49
34 Dry Cartridge (top) 10:49
35 Flush Carfridge Reagent Block 10:59
36 Flush Input Block 1 1 :09
37 Flush Small Loop (Carfridge) 11 :19
38 Load R3, Cartridge (small loop) 11:49
39 Transfer R3, Cartridge (gas) 11 :52
40 Wash Cartridge Solvent Block SI 11:57
41 Flush Cartridge Solvent Block 12:07
42 Flush Cartridge Reagent Block 12:17
43 Flush Input Block 12:27
44 Flush Small Loop (Carfridge) 12:37
45 Wash (Cartridge) Small loop SI 12:42
46 Flush Small Loop (Cartridge) 12:52
47 Flush Output Block 13:02
48 Wait 14:42
49 Dry Cartridge (top) 15:22 Step Function Name Elapsed Time
50 Ready Transfer to Flask 15:22
51 Flush Transfer Line 15:27
52 Del S3, Cartridge (sensor) 15:42
53 Cartridge Wait 16:02
54 Transfer to Flask (gas) 16:22
55 Flush Transfer Line 16:32
56 Del S2, Carfridge (sensor) 16:47
57 Carfridge Wait 17:07
58 Transfer to Flask (gas) 17:27
59 Flush Transfer Line 17:37
60 Del S3, Cartridge (sensor) 17:52
61 Cartridge Wait 18:02
62 Transfer to Flask (gas) 18:22
63 Flush Transfer Line 18:37
64 Transfer Complete 18:37
65 Del S3, Carfridge (top) 18:47
66 Cartridge Wait 18:57
67 Dry Cartridge (top) 19:57
68 End 19:57
RI 5% phenylisothiocyanate in hepane
R2 Methyl piperidine in n-propanol and water (25:60:15)
R3 Trifluoracetic acid (TFA)
R4 25% TFA in water
R5 PTH standard in acetonifrile (1 pmol/50 μl)
SI Heptane
S2 Ethyl acetate
S3 Butyl chloride
S4 7.5% acetonifrile in water X2 = Acetonifrile
X3 = Acetone
TABLE 3C
Method 1
Program Steps For A Rapid Edman Cycle
FLASK CYCLE
Step Function Name Elapsed Time
1 Begin :00
2 Set as Residue Cycle :00
3 Flush Small Loop (Flask) : 10
4 Load S4, Flask (small loop) :20
5 Dry Flask :30
6 Flush Small Loop (Flask) :40
7 Ready to Receive :41
8 Dry Flask :56
9 Pre-Conversion Dry 1 :21
10 Load R4, Flask (small loop) 1 :36
1 1 Dry Flask 1:46
12 Flush Small Loop (Flask) 1:56
13 Load S4, Flask (small loop) 2:06
14 Flush Small Loop (Flask) 2:16
15 Bubble Flask 2: 18
16 Wait 4:33
17 Bubble Flask 4:38
18 Wait 6:53
19 Bubble Flask 6:58
20 Post-Conversion Dry 8:16
21 Load Position 8:17 Step Function Name Elapsed Time
22 Prepare Pump 8:18
23 Dry Flask 9:18
24 Dry Flask 10:18
25 Dry Flask 13: 18
26 Flush Small Loop (Flask) 13:28
27 Load S4, Flask (small loop) 13:38
28 Dry Flask 13:48
29 Flush Small Loop (Flask) 13:58
30 Flush Large Loop (Flask) 14:13
31 Load S4, Flask (large loop) 14:33
32 Dry Flask 14:38
33 Flush Large Loop (Flask) 14:53
34 Flush Injector 15:23
35 Wait 15:28
36 Flush Injector 15:58
37 Dry Flask 16:03
38 Concentrate Sample 16:08
39 Wait 16:13
40 490A Relay 1 On 16:14
41 Load Injector 16:54
42 Inject Position 16:56
43 Start Gradient 16:56
44 490A Relay 1 Off 16:57
45 Wait 17:00
46 Del X2, Flask 17:20
47 Dry Flask 17:25 Step Function Name Elapsed Time
48 Bubble Flask 17:30
49 Flush Flask/Inject (High Pressure) 18:10
50 Empty Flask 18:20
51 Dry Flask 18:30
52 Flush Injector 18:50
53 Wait 19:50
54 End 19:50
RI = 5% phenylisothiocyanate in hepane
R2 = Methyl piperidine in n-propanol and water (25:60:15)
R3 = Trifluoracetic acid (TFA)
R4 = 25% TFA in water
R5 = PTH standard in acetonifrile (1 pmol/50 μl)
SI = Heptane
S2 = Ethyl acetate
S3 = Butyl chloride
S4 = 7.5% acetonifrile in water
X2 = Acetonifrile
X3 Acetone
TABLE 3D
Method 1
Program Steps For A Rapid Edman Cycle
FLASK STANDARD
Step Function Name Elapsed Time
1 Begin :00
2 Set as Standard Cycle :00
3 Del X2, Flask :20
4 Dry Flask :25
5 Empty Flask :45 Step Function Name Elapsed Time
6 Del R4, Waste :55
7 Flush Small Loop (Flask) 1:05
8 Flush Large Loop (Flask) 1 :20
9 Load R5, Flask (large loop) 1:40
10 Dry Flask 2:40
11 Load R4, Flask (small loop) 2:55
12 Dry Flask 3:05
13 Flush Small Loop (Flask) 3:15
14 Load S4, Flask (small loop) 3:25
15 Flush Small Loop (Flask) 3:35
16 Bubble Flask 3:37 7 Wait 5:52
18 Bubble Flask 5:57
19 Wait 8:12 0 Bubble Flask 8:17 1 Post-Conversion Dry 9:33 2 Load Position 9:34 3 Prepare Pump 9:35 4 Post-Conversion Dry 10:29 5 Dry Flask 14:29 6 Flush Small Loop (Flask) 14:39 7 Load S4, Flask (small loop) 14:49 8 Dry Flask 14:59 9 Flush Small Loop (Flask) 15:09 0 Flush Large Loop (Flask) 15:24 1 Load S4, Flask (large loop) 15:44 Step Function Name Elapsed Time
32 Dry Flask 15:54
33 Flush Large Loop (Flask) 16:04
34 Flush Injector 16:34
35 Wait 16:39
36 Flush Injector 17:09
37 Dry Flask 17:14
38 Concentrate Sample 17:19
39 Wait 17:24
40 490A Relay 1 On 17:24
41 Load Injector 18:04
42 Inject Position 18:05
43 Start Gradient 18:05
44 490A Relay 1 Off 18:05
45 Del X2, Flask 18:25
46 Dry Flask 18:30
47 Bubble Flask 18:35
48 Flush Flask/Inject (High Pressure) 19:15
49 Empty Flask 19:25
50 Dry Flask 19:45
51 Flush Injector 19:55
52 End 19:55
RI 5% phenylisothiocyanate in hepane
R2 Methyl piperidine in n-propanol and water (25:60: 15)
R3 Trifluoracetic acid (TFA)
R4 25% TFA in water
R5 PTH standard in acetonifrile (1 pmol/50 μl)
SI Heptane
S2 Ethyl acetate 53 = Butyl chloride
54 = 7.5% acetonifrile in water X2 = Acetonifrile
X3 = Acetone
Preferably, the "Begin," "Flask Std.," "Cartridge," and "Flask Residue" cycles are overlapped in time to maximize efficiency and sample 12 throughput. Thus, the Begin cycle may be run first on a first amino acid residue. Next, the Cartridge cycle is performed on the first amino acid residue, forming its ATZ derivative. Meanwhile, the Flask Std. is run as the Cartridge cycle is performed on the first amino acid residue. Next, during the Flask Residue Cycle, the ATZ derivative of the first amino acid residue is transferred to the conversion flask 17 and converted to the PTH derivative at the same time that a second amino acid residue is undergoing the coupling reaction in the Cartridge cycle. Finally, while a third amino acid residue is reacted in the Cartridge cycle, the second amino acid residue is converted in the Flask Residue cycle, and the first amino acid residue is analyzed as the PTH derivative on a chromatographic column. Each of the cycles is approximately 20 minutes or less, allowing the cycles to be conveniently overlapped in time for maximum time and cost savings.
The cycles described herein and in Table 3 may be performed using a commercial protein sequencing reagent delivery device such as, but not limited to, a PROCISE™ device, or its equivalent. Preferably, however, the samples 12 are reacted in the sample cartridges 20 of the apparatus 10 of the present invention. In addition, it is preferred that the apparatus 10 is operably coupled to the programmed Edman reaction cycles such that a different sample cartridge 20 is positioned for sample analysis upon completion of the predetermined number of amino acid residue analyses of the previous sample.
The "Begin" cycle (Table 3A) refers to an automated programmed cycle in which a protein sample is washed and a first coupling reaction is performed. This cycle is preferably performed once per sample as the beginning cycle to prepare the sample for further Edman cycles. More preferably, the Begin cycle is omitted since the coupling reactions of the "Cartridge" cycle are sufficient to achieve coupling of PITC to the N-terminal amino acid. The Begin cycle is an optional feature and is disclosed in Table 3A. Steps 1-13 of the Begin cycle perform valve and block washings with heptane (solvent SI).
Steps 14-23 perform a sample wash in which the solvents such as, but not limited to, ethyl acetate (solvent S2), butyl chloride (solvent S3), and acetone (wash X3) are used. Steps 24-38 perform the Edman coupling reaction in which PITC (PITC in heptane, reagent RI) is coupled to the N-terminal amino acid of the sample under basic conditions, such as in the presence of methyl piperidine in n- propanol and water (25:60: 15, designated reagent R2). Steps 39-56 perform a sample and block wash using heptane (block wash) and ethyl acetate, butyl chloride, and acetone (sample wash). The "Carfridge" cycle of Table 3B performs the coupling and cleavage reactions for each amino acid residue to be analyzed in the sample. The steps in the Cartridge cycle are preferably performed at 55 °C. Steps 1-16 perform the PITC coupling reaction by delivering 5% PITC in heptane (reagent RI, Table 3) to the sample in two portions under basic conditions in the presence of methyl piperidine in n-propanol and water (reagent R2, Table 3B). Steps 17-33 perform a sample wash using ethyl acetate (S2), acetone (X3), and butyl chloride (S3). Steps 34-48 perform cleavage and cyclization of the derivatized N-terminal amino acid to generate the ATZ-amino acid derivative under acidic aqueous conditions (such as in TFA (frifluoroacetic acid) in water, reagent R3, Table 3B). Steps 49-68 transfer the ATZ derivative to the conversion flask. The Flask Residue cycle of Table 3C is performed on a first amino acid derivative while the next amino acid residue in the sequence is coupled and cleaved in the Cartridge cycle. The steps in the Flask Residue cycle are performed in a conversion flask 17 in-line between the sample carfridge and the chromatographic column, a typical configuration for a protein sequencing device. The Flask Residue cycle is preferably performed at approximately 75 °C. Steps 1-7 prepare the conversion flask 17 and receive the ATZ derivative from the Cartridge cycle. Steps 8-19 convert the less thermodynamically stable ATZ-amino acid derivative to the more stable PTH-amino acid derivative under acidic conditions, such as in frifluoroacetic acid. Steps 20-38 dry the conversion flask 17 and then dissolve the PTH derivative in 7.5% acetonifrile in water. Steps 39-54 prepare the chromatographic injector and inject the PTH derivative onto an analysis system, such as a high pressure liquid chromatographic column for chromatographic analysis using the elution gradient shown in Table 1 A.
The user of the invention may perform an analysis of PTH-amino acid derivatives as necessary during the sample cycles. Preferably, a PTH-amino acid standard is run according to the Flask Std. cycle (Table 3D) performed simultaneously with the Begin cycle or the Carfridge cycle on the N- terminal amino acid of the sample. The steps of the Flask Std. cycle are preferably performed at 75 °C. Steps 1-5 of Table 3D wash the conversion flask with acetonifrile (X2, Table 3). Steps 6-10 deliver the PTH amino acid standard (R5) to the flask. Steps 11-20 allow the PTH amino acid standard to be exposed to the conditions of the conversion reaction. Steps 21-38 dry the conversion flask and deliver acetonifrile to dissolve the PTH amino acid standard. Steps 39-43 prepare the injector and inject the PTH amino acid standard onto the chromatographic column. Steps 44-52 wash the flask and injector in preparation for the next cycle.
Fast Edman Cycle Method 2:
Because some protein sequencing reagent delivery devices (such as the PROCISE 473A™ device) deliver a limited number of reagents, the following method, Method 2, is described to allow the fast Edman cycles of the method to be used with such a commercial device. Table 4 provides steps useful in practicing Method 2 of Rapid Edman Cycles, which method is compatible with a reagent delivery system for delivering fewer reagents and solvents than a system useful for Method 1. A regent delivery system useful in Method 2 may be, but is not limited to, a PROCISE 473™ reagent delivery system. As Table 4 shows, the Cartridge and Flask cycles are run simultaneously by a single program. The reagent and solvent designations used in Table 3 are also used in Table 4. Table 4. Method 2 of the Rapid Edman Cycle
CARTRIDGE CYCLE ELAPSED TIME
STEP # FLASK CYCLE (MIN:SEC)
1 Dry Cartridge Wait 00: 14
2 Dry Carfridge Load R4 00:28
3 Prepare R2 Dry Flask 00:34
4 Deliver R2 Load S4 00:43
5 Deliver R2 Block 45 Flush 00:48
6 Deliver R2 Wait 00:59
7 Prepare RI Wait 01 :04
8 Deliver RI Wait 01:05
9 Dry Cartridge Wait 01:20
10 Prepare R2 Wait 01:25
11 Deliver R2 Wait 03:30
12 Prepare RI Flask Bubble 03:35
13 Deliver RI Wait 03:36
14 Dry Carfridge Wait 03:51
15 Prepare R2 Wait 03:56
16 Deliver R2 Timed Dry 06:01
17 Dry Cartridge Flask Bubble 06:06
18 Dry Cartridge Wait 06:27
19 Dry Cartridge Timed Dry 07:47
20 Load Position Initialize Gradient 07:48
21 Dry Cartridge Timed Dry 08:03
22 Prepare S2 Timed Dry 08:08
23 Deliver S2 Timed Dry 08:20
24 Dry Cartridge Timed Dry 08:26
25 Deliver S2 Timed Dry 08:38
26 Deliver S2 Wait 08:43
27 Dry Carfridge Injector Clear 08:48
28 Deliver S2 Injector Clear 09:00
29 Dry Cartridge Valve Block 45 Flush 09:03 CARTRIDGE CYCLE ELAPSED TIME
STEP # FLASK CYCLE (MIN: SEC)
30 Dry Cartridge Valve Block 3 Flush 09:08
31 Dry Carfridge Flask Bubble, High Pressure 09:11
32 Dry Carfridge Load S4 09:20
33 Dry Carfridge Dry Flask 09:25
34 Dry Carfridge Load S4 09:34
35 Dry Cartridge Dry Flask 09:39
36 Dry Carfridge Valve Block 45 Flush 09:44
37 Dry Carfridge Wait 10:00
38 Prepare R3 Wait 10:05
39 R3 Gas Wait 10:08
40 R3 Gas Wait 10:11
41 R3 Gas Wait 10:21
42 R3 Gas Wait 11:25
43 R3 Gas Dry Flask 11:28
44 R3 Gas Wait 11:47
45 R3 Gas Dry Flask 11:50
46 R3 Gas Wait 12:10
47 R3 Gas High Pressure Injector Load 12:39
48 R3 Gas High Pressure Inject 12:41
49 R3 Gas Start Gradient 12:42
50 R3 Gas High Pressure Injector Clear 13: 12
51 R3 Gas Valve Block 45 Flush 13: 18
52 R3 Gas Flask Bubble, High Pressure 13:23
53 Dry Cartridge Deliver S4 13:43
54 Dry Cartridge Dry Flask, High Pressure 13:58
55 Dry Carfridge Empty 14: 18
56 Dry Carfridge Load S4 14:27
57 Dry Carfridge Dry Flask, High Pressure 14:47
58 Dry Cartridge High Pressure Injector Clear 15:02
59 Load S2 High Pressure Injector Clear 15:04
60 Block 1 Flush High Pressure Injector Clear 15:19
61 Wait Block 45 Flush 15:21
62 Wait Block 3 Flush 15:27
63 Prepare Transfer Load S4 15:36 CARTRIDGE CYCLE ELAPSED TIME
STEP # FLASK CYCLE (MIN:SEC)
64 Wait Dry Flask, High Pressure 15:41
65 Transfer S2 Flask Bubble 15:45
66 Wait Flask Bubble 15:55
67 Transfer Gas Flask Bubble 16:15
68 Transfer S2 Flask Bubble 16:20
69 Wait Flask Bubble 16:30
70 Transfer Gas Flask Bubble 16:50
71 Deliver S2 Dry Flask, High Pressure 17:05
72 Dry Cartridge Dry Flask 18:25
73 Dry Cartridge Timed Dry 20:05
RI = 5% phenylisothiocyanate in hepane
R2 = Methyl piperidine in n-propanol and water (25:60:15)
R3 = Trifluoracetic acid (TFA)
R4 = 25% TFA in water
R5 = PTH standard in acetonifrile (1 pmol/50 μl)
SI = Heptane
S2 = Ethyl acetate
S3 = Butyl chloride
S4 = 7.5% acetonifrile in water
X2 = Acetonifrile
X3 = Acetone
Steps 3-16 of Table 4 perform the PITC coupling reaction in the Carfridge cycle at 55 °C, while steps 1-19 perform the conversion of the ATZ-amino acid derivative to the PTH-amino acid derivative at 75 °C.
Step 20 directs the injector valve to be placed in the load position in the Cartridge cycle, while the chromatographic gradient is initialized by a command in the Conversion Flask cycle.
Steps 21-37 perform a solvent wash of the carfridge followed by drying, the conversion flask is also dried, and the injector is flushed and prepared to receive a converted amino acid derivative.
Steps 32-46 of the Conversion Flask cycle dissolve the PTH-amino acid derivative in 7.5% acetonifrile in water.
Steps 38-52 perform the cleavage reaction by delivering frifluoroacetic acid in gaseous form, thereby generating the ATZ-amino acid derivative. Steps 47-49 in the Conversion Flask cycle load the injector with the PTH-amino acid derivative and inject it onto the chromatographic column, followed quickly by the start of the chromatographic solvent gradient.
Steps 53-73 of the Cartridge cycle dry the cartridge, extract the ATZ-amino acid derivative into ethyl acetate, and transfer the extracted derivative to the conversion flask.
Steps 53-73 of the Conversion Flask cycle clear and dry the conversion flask for receipt of the ATZ-amino acid derivative from the carfridge, and clear the injector following the injection in Step 48.
Table 1A describes the chromatographic analysis useful in the invention following the fast Edman cycles of Methods 1 and 2. Example 3: Immobilization of Proteins onto PVDF Prior to Sequence Analysis.
This example describes a method of immobilizing proteins and polypeptides of interest onto a PVDF membrane prior to sequence analysis using the apparatus 10 of the invention with the rapid sequence analysis methods also provided herein.
Proteins for N-terminal sequencing were reduced in 20 μl of BioRad Laemmli sample buffer (BioRad, Richmond, CA), pH 8.3, 10 mM DTT, at 60°C for 15 minutes. Alkylation was performed by the addition of 0.08 mg of N-isopropyliodoacetamide according to the procedure of Krutzsch and
Inman (Krutzsch, H.C. and Inman, J.K., Anal. Biochem. 209: 109-116 (1993)) in 2 μl methanol at 25 °C for 20 minutes. Proteins were separated on precast gels (BioRad, supra) and electroblotted onto
Millipore Immobilon-PSQ™ or PE-Applied Biosystems Problott™ membranes in a BioRad Trans- Blot™ transfer cell using 10 mM CAPS (Sigma, St. Louis, MO) containing 10% methanol as the fransfer buffer at 250 mA constant current for 1 hour according to the procedure of Matsudaira
(Matsudaira, P., J. Biol. Chem. 262:10035-10038 (1987)). The PVDF membrane was stained with
0.1% Coomassie Blue R-250 in 50% methanol for 30 sec and destained with 10% acetic acid in 50% methanol for 2-3 minutes. The membrane was thoroughly washed with water and allowed to dry before optional storage at -20°C.
Prior to sequence analysis, portions of the PVDF membranes containing protein spots were cut to the approximate dimensions of 1 mm x 4 mm and inserted into a sample cartridge, such as a piece of Teflon™ tubing approximately 15 - 20 mm long, 1/16" i.d. and 1/8" o.d. Example 4: Automated Protein Sequencing. The present example demonstrates use of the apparatus 10 of the invention in which the sample cartridges 20 are contained in a stationary sample cartridge holder 22 and the reagent 30 delivery is controlled with a multi-position valve 43 as illustrated in Figure 1.
A PROCISE 473 A™ reagent delivery device 14 was in communication with a stationary carfridge holder 22 which holds the sample cartridges 20 in the horizontal orientation 80. Each sample cartridges 20 has a 1/16 inch internal diameter (i.d.) and a 1/8 inch outer diameter (o.d.). The length of each sample carfridge 20 was approximately 15 - 20 mm. The sample cartridges 20 were heated by a
4.6 cm x 7.6 cm Minco thermofoil heater (model HK913L, Minco, Minneapolis, Minnesota) attached to an aluminum holding block 70. According to one embodiment of the invention, carfridge selection was controlled by a 14-port stainless steel automated valve 43 (model EMT-CST6-UWTF, sold by Valco, Houston, TX). The valve 43 was equipped with a multi-position actuator control module (Valco model EMTCA) which was controlled by an external relay in the PROCISE 473A™ protein sequencer. Automated protein sequencing was performed on PE-Applied Biosystems protein reagent delivery devices, PROCISE 494A™ and a modified PROCISE 473A™. The PROCISE™ reagent delivery device was used with a high pressure liquid chromatographic system. The chromatography column 40, such as a 2.0 x 150 mm column by Alltech, (preferably PTH column part number C60003) was packed with 5 μm C18 resin (preferably PTH resin from Perkin Elmer- Applied Biosystems). An on-line PTH analyzer, such as a Perkin Elmer- Applied Biosystems 140C™ was also used. The coupling buffer was N-methylpiperdine in 1-propanol and water (25:60:15). For the chromatographic gradient of the analysis, solvent A comprised 3.5% THF in aqueous acetate buffer (such as 2% Premix™ Buffer Concentrate supplied by PE-Applied Biosystems). Chromatographic solvent B (Table 1A) comprised approximately 11% (optionally 12.5%) isopropanol in acetonifrile (Burdick and Jackson, HPLC grade). Optionally, solvent B may be acetonifrile without added isopropanol. Isopropanol was occasionally useful in enhancing the separation of the PTH-tryptophan derivative from phenylisothiocyanate. Further, acetone was routinely added to solvent A to balance the baseline. Peaks were integrated with Justice Innovation™ software using Nelson Analytical 760™ interfaces. Sequence interpretation was performed on a DEC Alpha™ computer according to the procedure described by Henzel et al. (Henzel, W.J. et al., J. Chromatogr. 404:41-52 (1987)).
Several proteins were identified by sequencing according to Method 1 of the rapid Edman cycle described herein. The N-terminal sequence of each protein and its identity is listed in Table 5. The term "Initial Yield" in Table 5 refers to the amount of protein or polypeptide sample at the beginning of a series of Edman cycles as determined by the calculated recovery of each amino acid residue in the series.
TABLE 5 Proteins Sequenced Using The Rapid Edman Cycle
Figure imgf000038_0001
* All Accession numbers are from Swiss Protein database except the persephin accession number which is from NCBI Example 5: Sorting Mixtures of Protein Sequences.
The SEQSORT algorithm was used to sort sequence mixtures (W. J. Henzel et al., "Analysis of Mixture Sequences Derived From Edman Degradation Data," in Techniques in Protein Chemistry, Marshak, D. R., ed., vol. VII, pp. 341-346 (1996)). The algorithm finds patterns specified as regular- expression syntax. It is similar in implementation to the UNIX regular-expression matching program (see UNIX User's Reference Manual (URM), 4.3 Berkeley Software Distribution Virtual VAX-1 1 Version, Computer Systems Research Group, Department of Electrical Engineering and Computer Science, University of California, Berkeley, California). SEQSORT has features beyond those of the UNIX regular-expression matching program. These additional SEQSORT features include (1) about 15 of allowed mismatches can be specified; (2) the search can be restricted to a region around the N- terminus; (3) the search can be limited to proteins with a specific molecular weight range; and (4) a species specific search can be performed. The algorithm begins by compiling the ambiguous sequencer data using a finite automaton to find regular expressions. The finite automaton described by Miller (Miller, W., "A Software Tools Sampler," Prentice-Hall, Englewood Cliffs, N.J. (1987)) was augmented by allowing transitions on mismatches, as long as the number of mismatches was below a user-specified threshold. Next, each sequence of the database was examined. Sequences having a molecular weight outside the specified limits were rejected. If the search was restricted to a region near the N-terminus, the sequence was truncated to the region of interest. A default value of 60 residues was typically used to allow a match with proteins that contained signal sequences that were usually less than 40 amino acids in length. The resulting sequence is checked for the existence of the specified pattern. If the pattern is found, the sequence is added to a list which can later be sorted by molecular weight or by the number of mismatches. When no amino acid was observed on a cycle, an "X" was specified, allowing any amino acid to match. The algorithm sorted a mixture of over 100 sequences. It is noted that as the number of sequences increases, the number of random matches also increases, requiring longer sequence analyses to prevent random matching. Example 6: Sequence Analysis of a Mixture of Proteins.
The single discreet bands observed following a one-dimensional SDS polyacrylamide gel separation of a mixture of proteins often contain several co-eluting proteins. The higher resolution of two-dimensional gel separations provides improved separation such that each spot frequently represents a single protein. Complete resolution, however, is not guaranteed, making a method that quickly and accurately identifies proteins within a mixture useful.
When a protein has been cleaved by proteolysis at several residues that are in close proximity to each other, the sequences that are obtained are often difficult to interpret. Although a match may be found from a database search, some cleavage sites may be missed. Complex mixtures arising from proteolysis can often be deciphered by searching the sequence mixture with the sequence of the protein only. Using this approach a larger number of mismatches can be used and the resulting matches can be sorted based on the quantitative Edman data.
A proteolytically cleaved protein (most likely a mixture) was observed as a single spot on a 2- D gel and was analyzed to determine the sequence of 10 amino acids from the existing N-terminus. A search of the protein sequence database, using this sequence of a potential mixture of proteins and the SEQSORT algorithm resulted in the identification of this sequence as intact apolipoprotein Cl and the cleaved form of this protein starting at residue 3.
When complex mixtures are analyzed, the possibility of false matches becomes significant. This problem can be minimized by (1) performing searches using longer sequences; (2) using a narrow molecular weight range; (3) matching within the N-terminal region of the protein only; and (4) using species specific searches.
Recombinant expression of IFN-alpha in E. coli resulted in a predominant band at approximately 19 l Da on a SDS-PAGE gel. Sequence analysis of this band failed to identify IFN- alpha. Three proteins were readily identified, however, using the SEQSORT algorithm with no false positives using an E. coli protein specific search (9134 proteins). Three E. coli proteins were found along with 13 false matches using the entire protein sequence database of 423, l 4 proteins. When 13 residues of sequence were included in the search instead of 12 residues, two false positive results were obtained. When the search was repeated using 12 residues of sequence and limited to a 10-30 kDa molecular weight range, only one false positive result was obtained. A search that used a match to the first 60 residues lead to the same false positive result (a viral protein).
Quantitative sequence analysis can lead to deciphering a mixture of sequences. This becomes difficult, however, when the protein concentrations in the mixture are similar. Residues typically exhibiting lower recovery (e.g., Ser, Thr, His, Arg, and Tip) can further complicate the analysis of a mixture based on PTH-amino acid yields. The SEQSORT algorithm was used without the requirement of quantitative data. When a match was found that satisfies all the properties of the proteins in the mixture, e.g., molecular weight, species, and isoelecfric point, then the PTH-amino acid yields can be utilized to further increase the confidence level of the database match. Figure 9 is a bar graph which illustrates the repetitive yield of three alternate protein accomplished using the apparatus 10 and methods of the present invention. Example 7: Describing Use Of The Modular Apparatus.
A module 220 including five apparatus sample cartridges 20, instead of the six sample cartridges 20 illustrated in Figure IB, was connected to be in fluid and electronic communication with a commercial sequencer 202, i.e., Procise 494™ reagent delivery device and protein sequencer. The module 200 was connected to the sequencer 202 in place of one of the sequencer sample cartridges 214. The module 200 was connected to the commercial sequencer, somewhat similar to that disclosed in Figure IB.
Automated protein sequencing was performed routinely using the module 200 and the methods described in Example 4. The module 200 effectively expanded a single sample position of the commercial sequencer 202 to five, sample positions. Expansion of each of the four single-sample positions of the sequencer 202 in this way expands the capacity of the commercial sequence 202 from four samples 12 to at least twenty samples 12. Further expansion is readily possible by increasing the number of apparatus sample cartridges 20 on each module 200. All references provided herein are hereby incorporated by reference in their entirety.
While the particular apparatus 10 as herein shown and disclosed in detail is fully capable of obtaining the objects and providing the advantages herein before stated, it is to be understood that it is merely illustrative of some of the presently preferred embodiments of the invention and that no limitations are intended to the details of construction or design herein shown other than as described in the appended claims.

Claims

What is claimed is:
1. An apparatus for use with a reagent delivery device and an analyzer for analyzing a plurality of samples, the reagent delivery device including a reagent delivery port for supplying one or more chemicals, the analyzer being adapted to analyze a residue formed from the interaction between one of the samples and one of the chemicals, the apparatus comprising: a plurality of apparatus sample cartridges, each apparatus sample cartridge defining a chamber sized and shaped to receive one of the samples, each apparatus sample cartridge including a cartridge inlet and a cartridge outlet; a cartridge holder including a plurality of spaced holding stations, each holding station being adapted to retain one of the apparatus sample cartridges in a substantially horizontal position so that the solvents flow substantially horizontally through the apparatus sample cartridge, each holding station including a heater for independently heating each apparatus sample carfridge to a predetermined temperature; and a fluid connector adapted to (i) independently connect the carfridge inlet of each apparatus sample carfridge in fluid communication with the reagent port so that the chemicals from the reagent delivery device can be selectively delivered to the apparatus sample cartridges and (ii) independently connect the carfridge outlet of each apparatus sample carfridge in fluid communication with the analyzer so that the residue in each apparatus sample carfridge can be delivered to the analyzer.
2. The apparatus of claim 1 wherein the fluid connector is a multi-port, radial valve.
3. The apparatus of claim 2 wherein the valve includes (i) a connector inlet port in fluid communication with the reagent port, (ii) a connector outlet port in fluid communication with the analyzer, (iii) a plurality of supply ports, each supply port being in fluid communication with one of the carfridge inlets and (iv) a plurality of return ports, each return port being in fluid communication with one of the cartridge outlets.
4. The apparatus of claim 3 further comprising an apparatus confroller electronically connected to the fluid connector for selectively establishing a path of fluid communication from the connector inlet port to one of the supply ports, the apparatus controller also being electrically connected to the reagent delivery device.
5. The apparatus of claim 4 further comprising a sensor which detects when one of the chemicals is positioned in one of the apparatus sample cartridges, the sensor being in elecfrical communication with the apparatus confroller so that information regarding whether the chemical is positioned in the apparatus sample cartridge can be transferred from the sensor to the apparatus controller and the reagent delivery device.
6. The apparatus of claim 4 wherein the apparatus controller operates the fluid connector according to an approximately twenty minute cycle.
7. A module including a module base and the apparatus of claim 1 secured to the module base.
8. A sequencer comprising (i) a sequencer reagent delivery device including at least two sequencer reagent ports, (ii) a sequencer analyzer including at least two sequencer analyzer ports, (iii) a sequencer sample carfridge in fluid communication with one of the sequencer reagent ports and one of the sequencer analyzer ports and (iv) the module of claim 7, wherein the fluid connector is in fluid communication with one of the sequencer reagent ports and one of the sequencer analyzer ports.
9. A module adapted for use with a sequencer for analyzing a plurality of samples, the sequencer including a sequencer sample cartridge, a sequencer reagent delivery device and a sequencer analyzer, the sequencer reagent delivery device being adapted to provide a plurality of chemicals to the sequencer sample cartridge through a sequencer reagent port, the sequencer analyzer being adapted to analyze a residue created by the interaction between one of the chemicals and the sample, the module comprising: a module base; a plurality of apparatus sample cartridges, each apparatus sample cartridge defining a chamber sized and shaped to receive one of the samples, each apparatus sample carfridge including a cartridge inlet and a cartridge outlet; a carfridge holder secured to the module base, the cartridge holder including a plurality of spaced apart holding stations, at least one of the holding stations being adapted to retain one of the apparatus sample cartridges in a substantially horizontal orientation so that the solvents flow substantially horizontally through the apparatus sample carfridge, at least one of the holding stations including a heater which individually heats one apparatus sample cartridge to a predetermined temperature; and a fluid connector secured to the module base, the fluid connector including a connector inlet port adapted to be in fluid communication with the sequencer reagent port and the fluid connector is adapted to independently connect the cartridge inlet of each apparatus sample cartridge in fluid communication with the connector inlet port so that the chemicals from the reagent delivery device can be selectively delivered to the apparatus sample cartridges.
10. The module of claim 9 wherein the fluid connector includes a connector outlet port adapted to be in fluid communication with a sequencer analyzer port of sequencer analyzer, and the fluid connector is adapted to independently connect the cartridge outlet of each apparatus sample cartridge in fluid communication with the connector outlet port so that a residue formed by the interaction between the chemicals and the sample in each apparatus sample carfridge can be delivered to the sequencer analyzer port.
11. The module of claim 10 wherein the fluid connector is a multi-port, radial valve.
12. The module of claim 11 wherein the valve includes (i) a plurality of supply ports, each supply port being in fluid communication with one of the cartridge inlets, and (ii) a plurality of return ports, each return port being in fluid communication with one of the cartridge outlets.
13. The module of claim 9 further comprising an apparatus confroller electronically connected to the fluid connector for controlling the fluid connector, the apparatus controller being adapted to be electrically connected to a sequencer confroller of the sequencer.
14. The module of claim 13 further comprising a sensor which detects when one of the chemicals is positioned in one of the apparatus sample cartridges, the sensor being in electrical communication with the apparatus controller so that information regarding whether the chemical is positioned in the apparatus sample cartridge can be fransferred from the sensor to the apparatus controller and the sequencer controller.
15. An apparatus for subjecting a plurality of samples to one or more chemicals from a reagent delivery device, the reagent delivery device including a reagent port, the apparatus comprising: a carfridge holder; a plurality of apparatus sample cartridges secured to the carfridge holder, each of the apparatus cartridges defining a chamber sized and shaped to receive one of the samples, each apparatus sample cartridge including a cartridge inlet and a cartridge outlet; a reagent outtake in fluid communication with the reagent port; a port holder which retains the reagent outtake; and a mover adapted to move one of the holders relative to the other holder so that the cartridge inlet of each of the apparatus sample cartridges is individually placed in fluid communication with the reagent outtake.
16. The apparatus of claim 15 including an analyzer intake retained by the port holder, the analyzer intake being placed in fluid communication with the cartridge outlet of one of the apparatus sample cartridges when the reagent outtake is in fluid communication with the cartridge inlet.
17. The apparatus of claim 15 wherein the mover is adapted to rotate the carfridge holder relative to the port holder.
18. The apparatus of claim 15 wherein the cartridge holder includes a plurality of holding stations, at least one of the holding stations including a heater adapted to individually heat the apparatus sample cartridge to a predetermined temperature.
19. The apparatus of claim 18 further comprising a source of electrical current which includes at least one source elecfrical contact; wherein the heater includes at least one heater elecfrical contact; wherein the mover moves the heater electrical contact relative to the source electrical contact until the heater electrical contact contacts the source electrical contact.
20. The apparatus of claim 15 including an analyzer in fluid communication with the analyzer intake, the analyzer being adapted to analyze a residue formed by the interaction between the chemicals and the sample in each sample carfridge.
21. The apparatus of claim 15 wherein the cartridge holder includes a plurality of spaced apart holding stations, each holding station being adapted to retain one of the apparatus sample cartridges in a substantially horizontal orientation so that the solvents flow substantially horizontally through the apparatus sample cartridges.
22. The apparatus of claim 15 further comprising an apparatus controller electronically controlling the mover.
23. The apparatus of claim 22 further comprising a sensor which detects when one of the chemicals is positioned in one of the apparatus sample cartridges, the sensor being in electrical communication with the apparatus controller so that information regarding whether the chemical is positioned in the apparatus sample cartridge can be fransferred from the sensor to the apparatus controller.
24 The apparatus of claim 22 wherein the apparatus controller operates the mover according to an approximately twenty minute cycle.
25. A method for analyzing a plurality of samples, the method comprising the steps of: providing a plurality of apparatus sample cartridges, each apparatus sample cartridge including a chamber, a carfridge inlet and a cartridge outlet; placing one sample in the chamber of each of the apparatus sample cartridges; holding the apparatus sample cartridges in a substantially horizontal orientation; independently connecting the carfridge inlet of each apparatus sample cartridge in fluid communication with a reagent port of a reagent delivery device so that the chemicals from the reagent delivery device can be selectively delivered to the apparatus sample cartridges; independently connecting the cartridge outlet of each apparatus sample carfridge in fluid communication with an analyzer port of an analyzer so that the residue formed by the interaction between the chemicals and the sample in each apparatus sample carfridge can be delivered to the analyzer port; and independently heating each apparatus sample cartridge to a predetermined temperature.
26. The method of claim 25 wherein the step of independently connecting the cartridge inlet includes the step of using a multi-port radial valve to individually connect the carfridge inlet of each apparatus sample cartridge in fluid communication with the reagent port.
27. The method of claim 25 when the step of independently connecting the cartridge inlet includes the step of moving the cartridge inlet of one particular apparatus sample cartridge, relative to the reagent port so that the reagent port is in fluid communication with the cartridge inlet of the one particular apparatus sample cartridge.
28. A method for increasing the throughput of a sequencer, the sequencer including a sequencer sample cartridge, a sequencer reagent delivery device and a sequencer analyzer, the sequencer reagent delivery device being adapted to provide a plurality of chemicals to the sequencer sample carfridge through a sequencer reagent port, the sequencer analyzer being adapted to analyze a residue created by the interaction between one of the chemicals and the sample, the method comprising the steps of: providing a module including (i) a module base, (ii) a cartridge holder secured to the module base, (iii) a plurality of apparatus sample cartridges secured to the carfridge holder, each apparatus sample cartridge defining a chamber sized and shaped to receive one of the samples, each apparatus sample cartridge including a cartridge inlet and a carfridge outlet, and (iv) a fluid connector secured to the module base, the fluid connector including a connector inlet port and a connector outlet port, and the fluid connector being adapted to independently connect the carfridge inlet of each apparatus sample carfridge in fluid communication with the connector inlet port and independently connect the cartridge outlet of each apparatus sample carfridge in fluid communication with the connector outlet port; placing the sequencer reagent port in fluid communication with connector inlet port of the fluid connector; and placing the sequencer analyzer in fluid communication with connector outlet port of the fluid connector.
29. The method of claim 28 including the steps providing a plurality of modules, placing the sequencer reagent port in fluid communication with the connector inlet port of each module and placing the sequencer analyzer in fluid communication with the connector outlet port of each module.
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