Multidimensional pump apparatus and method for fully automated complex mixtures separation, identification, and quantification
A fluid pumping system can be operated in dual control modes: as a syringe pump for nano-flow solvent delivery; and as a reciprocating pump for micro- and analytical flow solvent delivery. The fluid pumping system is also operated in a closed-loop digital control process using an optical encoder for piston refill stroke begin and end synchronization with a switching valve On and Off. A multidimensional apparatus and procedure using up to 8 of the invented fluid pumps for a fully automated procedure such as ICA™ chemistry for cellular protein separation, identification and quantification.
1. Field:
The invention relates to high-pressure fluid pumping system that is capable of fluid delivery from nano- to analytical flow rates and more particularly to multidimensional HPLC instrumentation and method for the separation, identification, and quantification of complex mixtures such as cellular proteins and carbohydrates.
2. State of the Art:
Fluid pumping systems for high-pressure liquid chromatography (referred to hereinafter as HPLC) and the like are well know. In HPLC, complex sample with multiple components is transported through a column packed with particles of designed selectivity to cause separation of the components of the mixture when mobile solvent that is pumped through the column transports them. Typical prior art pumps employed in these applications were exemplified in U.S. Pat. No. 4,045,343 to Achener et al, U.S. Pat. No. 4,260,342 to Leka et al, U.S. Pat. No. 3,599,045 to Gordon et al, and U.S. Pat. Nos. 5,253,981, 5,664,938, and 5,630706 to Yang et al. The micro-flow fluid pumping system invented by Yang et al is particularly important today for application in micro-LC-mass spectrometry (micro-LC-MS) in the separation and identification of biological samples. Today, the need for even lower flow rate fluid delivery at nano-liter/min range is emerging as the analysis of low abundance biological molecules such as cellular proteins and carbohydrates is growing in interest and important. Several LC instrument manufacturers have developed micro-HPLC pumping systems. The typical commercially available micro-HPLC systems are modified versions of conventional low pressure proportioning HPLC gradient pumps. See H. Bente, et al. U.S. Pat. No. 4,714,545; G. Leka al, U.S. Pat. No. 4,260,342; P. Trafford et al, U.S. Pat. No. 4,728,434; P. Achener, et al, U.S. Pat. No. 4,045,343; J. Rock, U.S. Pat. No. 4,128,476; H. Magnussen, Jr. U.S. Pat. No. 4,180,375; H. Magnussen, Jr, U.S. Pat. No. 4,131,393; and R. Allington, U.S. Pat. No. 4,869,374. Such conventional systems use the split-flow technique (Sj. van der Wal et al., J. High Resolut Chromatogr. Comm. 6: 216, 1983), to obtain nl/min for nano-LC applications. Unfortunately, such split flow techniques are not reliable for routine applications at nano-flow rates as the flow restriction through the capillary column could change due to column inlet port blockage or flow restriction change inside the flow splitter. It is particularly difficult to maintain a constant split ratio when a high split ratio (for example 500 μl/min to 0.1 μl/min split i.e. 5000 to 1 split ratio) is required for nano-flow rate applications. An alternate approach for pumping in micro-HPLC is the single-stroke syringe-type piston pump (M. Munk, U.S. Pat. No. 4.032,445; R. Brownlee, U.S. Pat. No. 4,347,131; and R. Allington, U.S. Pat. No. 4,775,481. This type of syringe pump is capable of delivering solvent at a few μl/min. as limited by the number of steps per revolution of the stepping motor for driving the syringe piston. None of the existing HPLC apparatus can be used for flow rate ranges desirable in both splitless Nano/capillary column-HPLC at a flow rate range of 1 nl/min to 10 μl/min and micro/analytical column-HPLC at a flow rate range of 10 μl/min to 1,500 μl/min. This invention addresses a pumping system that has dual flow rate delivery ranges without flow splitting and can perform both nano/capillary column-HPLC in a flow rate range between 1 nl/min to 10 μl/min as well as micro/analytical-HPLC in a flow rate range between 1 μl/min to 1,500 μl/min for separation, identification, and quantification of low abundance biological samples such as cellular proteins and proteins reaction products. An example for the application of the nano-HPLC and Micro-HPLC dual flow rate ranges of this invention is shown for its usage in a fully automated on-line multi-dimensional HPLC-ICAT(Isotope-Coded Affinity Tag, (ICAT) Aebersold, R Gugi et al. Amercan Genomic Proteomic Technology, July/August 2001, 22-27) for cellular proteins separation, identification, and quantification.
As the inherent complexity of cellular proteins could be as much as several thousands and with a wide dynamic difference in abundance, it is necessary to develop the separation technique for rapid separation, identification, and quantification of those biological samples. The 2-Dimensional Gel Electrophoresis followed by direct analysis by tandem mass spectrometry of peptide mixtures generated by the digestion of complex protein mixtures has been proposed (Dongre A. R. et al., Trend Biotechnol. 1977 15: 418-425). However, the 2-D gel electrophoresis method is difficult to automate and cannot detect low abundance proteins. Micro-LC-MS/MS has also been used successfully for separation and identification of proteins without gel electrophoresis separation (Opitek G. J. et al., Anal. Chem. 1997 69: 1518-1524). With the commercialization of capillary column HPLC and electrical spray/nano-spray interfaces for mass spectrometers, the interest for finding cancer cellular protein markers is growing significantly in the past four years. The invention of Goodlett et al (U.S. Pat. No. 6,629,040) provides method and reagents for cellular proteins analysis. The method utilizes affinity-labeled protein reactive reagents that allow for selective isolation of peptide fragments or products of reaction with a given protein from complex biological mixtures. Isolated peptides or reaction products are identified by mass spectrometer (MS). In particular, the sequence of isolated peptides can be determined using tandem MS techniques, and by applications of sequence database searching techniques, the protein from which the sequenced peptide originated can be identified. The reagents also provide for differential isotopic labeling of the isolated peptides or reaction products, which facilitates quantitative determination by mass spectrometry of the relative amounts of proteins in different samples. Also, the use of differentially isotope labeled reagents as internal standards facilitates quantitative determination of the absolute amounts of one or more proteins present in the sample.
The Isotope-Coded Affinity Tag (ICAT) chemistry as presented by Goodlett et al (U.S. Pat. No.: 6,629,040) requires multiple off-line manual reaction processes. It cannot be practiced with small quantity of samples and is not automated. As a result, the method was not easily applicable to routine low abundance cellular proteins separation, identification, and quantification. This invention is to provide a fully automated multidimensional apparatus that allows on-line complex sample separation, reaction, cleavage, and nano-LC-MS-MS procedures. More specifically, the invention provides fully automated steps that utilizes multi-dimensional HPLC instrument and switching valves to fully automated ICATT™ (S. P. Gygi et al Nat. Biotechnol. 1999, 17, 994-999), IMAC (Immobilized metal affinity chromatography (IMAC) (L. Andersson et al Anal biochem. 1986, 154(10), 250-254) multi-steps procedures and to allow reproducible multi-dimensional capillary/micro-bore column HPLC and mass spectrometry analysis of low abundance cellular proteins and protein reaction products.
SUMMARY OF THE INVENTIONThe apparatus of the present invention comprises five elements. The first element is the means for pumping liquid such as a HPLC that is designed to allow pumping capability from 1 nl/min to 1,500 μl/min with or without flow splitting to ensure reliable and reproducible operations to meet the requirement of proteome analysis for clinical or diagnostic applications. The fluid pumping has high-speed two positions 6 port valve to allow reliable fluid channel switching to synchronize with the piston begin and end of a refill stroke. The second element is the combination of at least six or more of the first elements and reagents with valve switching capability for fully automated on-line ICAT™ or IMAC procedures. The third element is the utilization of multi-channel selection valves for parallel processing of multiple sample streams for sample trapping or collection. The forth element is the utilization of ion-exchange columns, reversed phase HPLC columns, affinity HPLC columns, size exclusion columns, metal chelating columns, Biotin cleavage columns, capillary reversed phase columns in series or in its combination to facilitated on-line ICAT, Immobilized metal affinity chromatography ((IMAC)Andersson, L; Anal. Biochem. 1986, 154 (1) 250-254), or other multiple complex sample analysis procedures that require more than three separation means or procedures. The fifth element is the use of tandem mass spectrometry and software for protein identification and quantification.
An example for the applications of the apparatus of the present invention is details in the following 12 steps procedures for ICAT™ or IMAC applications. The first step is to transport isotope labeled protein digest sample by pump 17 of the first element from an auto-sampler 25 or a HPLC injection valve 42 into an ion exchange 30 or size exclusion column. The second step is to stepwise or linearly increase the composition ratio of pump 18 (Salt) to pump 17 (H2O) of the first element for selectively eluting a portion of protein digest sample from the SCX column 30 into the trap column 31. The step 3 is to pump reagent A (PH 7) by pump 19 of the first element to neutralize the trapping column 31. The step 4 is to switch valve 3 to allow the condition of the Avidin affinity column 3 to PH=7.2 with pump 19 of the first element. The step five is to elute Biotin labeled protein digest by pumping elution reagent using pump 20 of the first element into the Avidin affinity column 32. The step 6 is to elute non-cystein containing peptides to collection vial A from the avidin column 32 using pump 21 of the first element. The step 7 is to elute biotin labeled cystein-containing peptides into the cleavage column 33 by using pump 22 of the first element. The step 8 is to transport cleavage reagents A and B that is mixed in the auto-sampler 25 into the cleavage column 33 by using pump 17 of the first element. The step 9 is to cleave the biotin from cystein containing peptides in the cleavage column 33 at 37° C. for 2 hours. The step 10 is the step while cleavage column 33 is in the cleavage step, the pump 17 of the first element is transporting the cleaved biotin to the waste 42 from cleavage column 33 that had completed steps 1 through 9 and ready for nano-LC-MS-MS. The step 11 is the step where the nano-LC-MS-MS at 50 to 2000 nl/min solvent gradient flow rate with or without flow splitting was delivered by pump 23 and 24 of the first element to allow cystein containing isotope labeled peptides to be separated, identified and quantified by the nano-LC-MS-MS technique with the nano-trap column 35 and nano-LC column 36 in the preferred configuration as showed in the
In the above step one; a high-pressure injection valve can be used to inject sample. The cleavage reagents A and B in the step 8 can also be pumped by two syringe pumps into a micro-mixer and then to the cleavage column(s). A multi-channel selection valve 8 (e.g. Valco 24 port multichannel selection valve) can be utilized to reduce the total analysis time of a complex sample. The apparatus includes the “multi-channel selection valve” as shown in
Referring to
Although the multi-dimensional pumping system and the closed loop digitally controlled gear motor driven pump are described primarily with reference to HPLC and ICAT™ analysis of cellular proteins, their uses are not limited to HPLC and ICAT™ applications (S. P. Gygi et al Nat. Biotechnol. 1999, 17, 994-999). The invention may include applications in proteome analysis, carbohydrates analysis, phosphoprotein isotope-coded affinity tag chemistry (M. B. Goshe et al, Anal Chem. 2001, 73, 2578-2586), Immobilized metal affinity chromatography (IMAC) (L. Andersson et al Anal biochem. 1986, 154(10), 250-254), and Multidimensional protein identification technology (MudPIT)(A. J. Link et al Nat. Biotechnol. 1999, 17, 676, W. H. McDonold et al Intern. J. Mass Spec 2002, 219, 245-251), capillary eletrophoresis, or other technology where multiple high pressure fluid delivery and valve switching and/or accurate, nano-flow rates, micro-flow rates, and analytical flow rates fluid delivery are necessary.
Claims
1. A fluid pump containing an optical encoder to facilitate digital closed loop control for synchronizing piston refill begin and end strokes with the opening and closing of electrically actuated or pneumatically actuated valve or valves switching.
2. The fluid pumping system of claim 1 wherein said the piston stroke length is adjustable and the piston refill stroke start and stop for the given length is in synchronization with switching valve open and close to solvent reservoir.
3. The fluid pumping system of claim 1 contains a Servomotor, a gear motor, a stepping motor, or a combination of motors and gearboxes with an optical encoder.
4. The fluid pumping system of claim 1 wherein said the pump can be operated as a syringe pump or a reciprocating pump by using a closed loop digital controlled by a computer software or firmware operation system.
5. The fluid pumping system of claim 1 contains a flex coupler to connect the motor to a Linear Actuator to facilitate linear driving of a piston that is mounted securely to the piston holder mounted on the actuators moving plate form.
6. A method of proportioning fluid to form a fluid composition or flow gradient, comprising the steps of:
- Providing a plurality of pumps each connected to a respective fluid from a corresponding one of a plurality of reservoirs or each connected to a plurality of reservoirs;
- Providing a plurality of switching valves each connected to a column or columns for on-line fluidic switching;
- Providing a plurality of separation columns to facilitate a separation of complex sample or samples based on a combination of more than two of the following separation methods: reversed phase, normal phase, ion exchange, size exclusion, affinity chromatography, gel permeation chromatography, electrical field assisted PH gradient chiral chromatography, metal chelating or affinity, isotope labeling tag, super-critical fluid chromatography, high temperature liquid chromatography, capillary electrophoresis(CE), capillary electro-chromatography(CEC), displacement chromatography, perfusion chromatography, and turbulence flow chromatography;
- A gradient proportioning pumping system for on-line fully automated separation of proteins or the reaction products of proteins. An example based on the Isotope-Coded Affinity Tag (ICAT™) chemistry as presented by Goodlett et al (U.S. Pat. No.: 6,629,040), or based on IMAC (Immobilized metal affinity chromatography (IMAC) (L. Anderson et al Anal biochem. 1986, 154(10), 250-254), comprising:
- A multi-channel pumping system including Six to eight pumps for a plurality of solvents; a strong cation exchange column, a reversed phase trapping column, an affinity column (Avidin affinity column), a cleavage column or multiple of cleavage columns, a nano-trap column, an analysis nano-LC column, a Mass Spectrometer, an auto-sampler, an injection valve or switching valve, three to four additional flow path diverting valves, one to two Tees, a spray tip or a nano-column with spray tip for Mass Spectrometer, and the Reagents prescribed in the Applied Biosystems ICAT™ kit (Applied Biosystems ICAT™ Cleavable ICAT™ Reagent Methods Development Kit, Part number 4339035).
7. The switching valve of the claim 6 includes also a multiple channel selection valves for on-line multiple sample parallel cleavage operations or sample collections
8. The auto-sampler injection valve of the claim 6 includes also a manual or time programmable injection valve;
9. A method for on-line automated ICAT™ protein or protein reaction products separation, identification, and quantification comprising
- Step 1: Sample injection into an ion exchange (SCX) column
- Step 2: By controlling composition ratio of elution strength of solvent, the sample trapped in the ion exchange column can be either partially eluted in a controlled fraction or completely eluted from the ion exchange column.
- Step 3: for removing salt and to condition the trapping column to PH=7.2
- step 4: for conditioning the Avidin column to PH=7.2.
- step 5: for neutralizing the ion exchange and Avidin columns after sample loading.
- step 6: for removing non-Biotin labeled peptides
- step 7: for eluting labeled cystein containing peptides from the Avidin column into the cleavage column. A 10-port or more than 10 port selection valve is used for switching the flow paths to a array of collection vials, plates, or cleavage columns for further processing.
- step 8: for pumping the cleavage reagent A and B that is freshly prepared by the auto-sampler into the cleavage column at 37° C. for Biotin cleavage of the cystein containing peptides.
- step 9: for connecting the cleavage column, an electrode, and a pre-column to the first Tee or cross. The pre-column is then connected to a second Tee where the analysis nano-LC column and a waste solvent transfer line are also connected. The outlet of the solvent waste transfer line is connected to a 6 port or a 4 port-switching valve for flow stream diversion. In this particular arrangement peptides of interest are trapped and concentrated at the pre-column and sample solvent goes to waste.
- step 10. Wherein the sample is chromatographically separated using nano-LC or capillary-LC binary gradient in both the pre-column and the analysis nano-LC column and then is sprayed into mass spectrometer ionization source for identification and quantification. The nano-LC fluid pumping system of claim 1 in splitless mode at 0.1 to 1.0 μl/min flow rates is a particularly useful because of its nano-flow gradient capability.
- Step 11: allows the sample in the ion exchange column is again eluted into the trap column to repeat those steps 1 through 10 again until the whole peptide sample in the ion exchange column has been completely eluted and analyzed;
10. A gradient proportioning pumping system of claim 6 for on-line fully automated separation of proteins or protein reaction products in proteome analysis, carbohydrates analysis, phosphoprotein isotope-coded affinity tag chemistry (M. B. Goshe et al, Anal Chem. 2001, 73, 2578-2586), Immobilized metal affinity chromatography (IMAC) (L. Andersson et al Anal biochem. 1986, 154(10), 250-254), and Multidimensional protein identification technology (MudPIT)(A. J. Link et al Nat. Biotechnol. 1999, 17, 676, W. H. McDonold et al Intern. J. Mass Spec 2002, 219, 245-251), capillary eletrophoresis, or other technology where multiple high pressure fluid delivery and valve switching, nano-flow rates, micro-flow rates, and analytical flow rates fluid delivery are necessary.
Type: Application
Filed: Mar 29, 2004
Publication Date: Sep 29, 2005
Inventor: Frank Yang (Vista, CA)
Application Number: 10/812,575