WO2006011351A1 - Absolute quantitation of protein contents based on exponentially modified protein abundance index by mass spectrometry - Google Patents
Absolute quantitation of protein contents based on exponentially modified protein abundance index by mass spectrometry Download PDFInfo
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- WO2006011351A1 WO2006011351A1 PCT/JP2005/012705 JP2005012705W WO2006011351A1 WO 2006011351 A1 WO2006011351 A1 WO 2006011351A1 JP 2005012705 W JP2005012705 W JP 2005012705W WO 2006011351 A1 WO2006011351 A1 WO 2006011351A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Definitions
- the present invention generally relates to analysis of protein contents by means of mass spectrometry, and more particularly to a method and a computer program for executing quantitation of the protein contents based on protein abundance index by mass spectrometry in proteomics.
- LC-MS Proteomic liquid chromatography-mass spectrometry
- protein concentrations in a sample can be also used for relative quantitation between two samples even when the difference in concentration is too large to perform isotope-based relative ⁇ quantitation.
- isotope-labeled synthetic peptides were used as internal standards for absolute quantitation of particular proteins of interest [11 ,12]. This approach would be applicable to comprehensive analysis but the cost of isotope-labeled peptides as well as the difficulty to do quantitative digestion of proteins in gel would cause a problem [13].
- the present inventor explores the PAI strategy to determine protein abundance from nanoLC-MS/MS experiments. It is an object of the present invention to provide a method for executing quantitation of protein contents based on an exponentially modified PAI (hereinafter referred as to "EMPAI") in a sample of biological material.
- EMPI exponentially modified PAI
- a computer program product for example, a computer readable medium which can be read by a computer and stores a computer program for executing quantitation of protein contents based on the above EMPAI in a sample of biological material.
- a computer program for executing quantitation of protein contents based on the above EMPAI in a sample of biological material there is also provided.
- an analytical apparatus for executing quantitation of protein contents based on the above EMPAI in a sample of biological material.
- the present invention provides a method for executing quantitation of protein content in a sample of biological material, said method comprising the steps of: (a) identifying a protein to be quantified by mass spectrometry; (b) measuring the number of observed peptides per the protein (Nobsd); (c) calculating the number of observable peptides per protein (Nobsbi); and (d) computing the following equation to obtain EMPAI:
- EMPAI 10 Nobsd/Nobsbl - 1.
- the method further comprises calculating protein contents (mol%) based on a value of EMPAI as follows:
- the method further comprises calculating protein contents (weight%) based on a value of EMPAI as follows:
- the mass spectrometry comprises a liquid chromatography-mass spectrometry.
- the present invention also provides a computer program product for executing quantitation of protein content in a sample of biological material, said program product comprising: a computer readable storage medium having a computer program stored there on for performing the steps: (a) identifying a protein to be quantified by mass spectrometry; (b) measuring the number of observed peptides per the protein (N O bsd); (c) calculating the number of observable peptides per protein (No b sbi); and (d) computing the following equation to obtain EMPAI
- the program comprises performing of calculating protein contents (mol%) based on a value of EMPAI as follows:
- the program comprises performing of calculating protein contents (weight%) based on a value of EMPAI as follows:
- ⁇ (EMPAI) is the summation of EMPAI values for all identified proteins and MW represents molecular weight of each protein identified.
- the product is a computer readable recording medium which can be read by a computer.
- the present invention also provides a computer program which executes quantitation of protein content in a sample of biological material, said program comprising performing the steps of: (a) identifying a protein to be quantified by mass spectrometry; (b) measuring the number of observed peptides per the protein (Nobsd); (c) calculating the number of observable peptides per protein (N Ob sbi); and (d) computing the following equation to obtain EMPAI
- the program further comprises performing of calculating protein contents (mol%) based on a value of EMPAI as follows:
- the computer program according to the present invention is characterized in that this program causes the respective steps of the method for quantifying protein content according to the present invention to be performed by a computer.
- the computer program can also be provided in the form of a storage medium where the program is stored as well as can be supplied via a transmission medium, such as the Internet.
- the present invention also provides an analytical apparatus for executing quantification of protein content in a sample of biological material, comprising: identifying means for receiving information as to a mass spectrometric data of proteins obtained by mass spectrometry and identifying a protein to be quantified by the mass spectrometry; measuring means for measuring the number of observed peptides per the protein (N Ob sd); calculating means for calculating the number of observable peptides per protein (Nobsbi); and computing means for computing the following equation to obtain EMPAI
- EMPAI 10 Nobsd/Nobsbl - 1 .
- ⁇ (EMPAI) is the summation of EMPAI values for all identified proteins.
- the mass spectrometry comprises a liquid chromatography-mass spectrometry.
- An advantage of the present invention is that the scale for absolute protein abundance, namely exponentially modified protein abundance index is established, which can use for absolute quantitation of protein contents in proteomics.
- Figure 1 shows a drawing of the hardware structure for the computer executing quantitation of protein contents based on the above EMPAI according to the present invention
- Figure 2 shows a block diagram which is used to illustrate the construction of an analytical apparatus for executing quantitation of protein contents based on the EMPAI according to the present invention
- Figure 3 shows a flowchart for executing quantitation of protein contents based on the EMPAl according to the present invention
- Fig. 4 shows one example of a flowchart for calculating the number of observable peptides per the protein;
- Figure 5 illustrates dependence of the number of peptides and peak area on the injected amounts of human serum albumin (HSA).
- Figure 5A shows peak area and the number of unique parent ions of peptides versus injection amounts of (HSA).
- Fig. 5B shows three different numbers of peptides versus injection amounts of HSA;
- Figure 6 shows the relationship between protein concentration and different parameters for 47 proteins in neuro2a cells.
- Figure 6A shows protein concentrations versus PAI.
- Figure 6 B shows protein concentration versus the number of peptides divided by molecular weight of proteins.
- Figure 6C shows protein concentration versus Mascot score.
- Figure 6D shows protein concentration versus the number of observed peptides (unique parent ions);
- Figure 7 shows the influence of MS measurement conditions on linear relationship between PAI and log[protein].
- Figure 7A shows time of fright type mass spectrometry (QSTAR) with slower scans.
- Figure 7B shows ion trap type mass spectrometry (LCQ) with slower scans;
- Figure 8 shows the relationship between protein concentrations and EMPAI for 47 proteins in neuro2a cells;
- FIG. 9 shows the results of absolute quantitation of 47 proteins in neuro2a using EMPAI according to the present invention.
- Figure 10 shows the comparison between gene and protein expression in HCT116 cells according to one embodiment of the present invention.
- FIG. 1 shows a drawing of the hardware structure for the computer carrying out quantitation of protein contents based on the above EMPAI by the LC-MS according to the present invention.
- An analytical apparatus 10 for executing quantitation of protein contents based on the EMPAI according to the present invention comprises a central processing unit 12 (hereinafter abbreviated as "CPU"), a memory 14, a display device 16, a user interface 18, and a communication interface 22, all mutually connected via a bus 24 with the CPU 12.
- the apparatus 10 further comprises an external storage (not shown in FIG. 1), such as a CD-ROM or a magnetic medium, connected to an external storage medium drive unite 20.
- the apparatus 10 can be connected to the external data base, such as NCBInr (hhtp://www.ncbi. nlm.nih.gov/) and so on, through the communication interface 22.
- the apparatus 10 can also be connected to the mass spectrometric device via the communication interface 22, which carries out analysis of the proteins.
- Fig. 2 shows a block diagram which is used to illustrate the construction of the analytical apparatus 10 for executing quantitation of protein contents based on the EMPAI according to the present invention.
- the apparatus 10 comprises IF (interface) means 30 and control means 40.
- the apparatus 10 is constructed so that these means 30, 40 receive input, for example, a mass spectrometric data, from the user utilizing this apparatus and/or a mass spectrometry, and output information to this user and/or the mass spectrometry.
- An ordinary personal computer can be used as the apparatus 10.
- Examples of the mass spectrometric data include a mass spectrum, a mass chromatogram and MSMS data and so on.
- the IF means 30 is constructed so that information can be input and output with respect to input device such as a keyboard, the mass spectrometry or the like and output device such as a display, printer or the like. Via the IF means 30, the mass spectrometric data to be analyzed is transmitted to the control means 40.
- the control means 40 comprises identifying means 42, measuring means 44, calculating means 46 and computing means 48.
- the identifying means 42 can receive information as to mass spectrometric data of proteins via the IF means 30 and identifies a protein to be quantified by mass spectrometry.
- the measuring means 44 measures the number of observed peptides per the protein (No bsd ) which has been identified by the identifying means 42.
- calculating means 46 calculates the number of observable peptides per protein (No bsb i).
- the term “the number of observed peptides per protein” used herein means that the number of peptides per protein to be quantified which was actually observed by the mass spectrometry.
- the term “the number of observable peptides per protein” used herein means that a theoretical number of peptides per the protein. It should be noted that these numbers are defined in the document [17].
- the computing means 48 which receives information as to N O bsd and Nobs b i, computes the following equation to obtain EMPAI:
- the computing means 48 calculates protein contents (mol%) and protein contents (weight %) in accordance with the two equations as follows:
- the computed and/or calculated data can be stored in memory means, which is not shown in Fig. 2.
- Fig. 3 shows a flowchart for executing quantitation of protein contents based on the EMPAI according to the present invention.
- a protein of interest is identified by the identifying means 42 after performing the mass spectrometry of samples of the biological materials by a MS method.
- This MS method includes peptide mass finger printing method and MS/MS method. It is understood by those skilled in the art that as disclosed in the following documents (Proc. Natl. Acad. Sd. USA. 1993, 90, 5011-5015, J. Curr. Biol. 1993, 3, 327-332, Biol. Mass. Spectrom. 1993, 22, 338-345, Nat Genet. 1998: 20, 46-50; J Cell Biol.
- step S11 the number of the observed peptides per the protein identified above is measured by the measuring means 44 with use of the MS data. Then, the number of observable peptides per the protein is calculated by the calculating means 46 based on the structure of the protein identified above (as shown in step S12). It is possible to calculate the number of observable peptides per the protein prior to measurement of the observed peptides per the protein.
- Fig. 4 shows one example of a flowchart for calculating the number of observable peptides per the protein, which is used in the present invention.
- the mass range is determined by using the observed peptides and the scan range of mass spec.
- the predicted retention times in each observed peptides are calculated based on Meek's equation (as described in [23]). Note that amino acid sequence of each observed peptides can be determined, for example, by the MS/MS method and according to the Meek's equation, there is the relationship between the known amino acid sequence and the retention time in liquid chromatography.
- the predicted retention time range is determined by using a retention time of observed peptides, based on the above Meek's equation.
- the digested tryptic peptides without missed cleavage are calculated by in silico (in step S124). More specifically, since trypsin is famous protease by which the peptide bond can selectively be cleaved at the carboxylic side of lysine residue and arginine residue in the protein, there is determined amino acid sequences of the digested tryptic peptides without missed cleavage. Thus, in this step S124, molecular weight (MW) and predicted retention time of the digested tryptic peptides are calculated in silico.
- the observable peptides are sorted according to the MW and the predicted retention time.
- the number of observable peptides per protein is counted, on the basis that MW and the predicted retention time of the observable peptides fall both within the mass range (in S121 ) and retention time range (in S123).
- MW is the molecular weight of each protein identified
- ⁇ (EMPAI) is the summation of EMPAI values for all identified proteins (as shown in Steps 14 and 15).
- a program which executes the flow of the analysis of protein contents illustrated in FIG. 3, which is discussed above, is stored in the memory 14 or the external storage via the external storage medium drive unit 20 or is directly transferred to the CPU 12 in case where the program is stored in the external storage medium, such as the CD-ROM.
- the protein is identified by the reference to the external database, such as NCBInr database, through the communication interface 22.
- the display device 16 displays results of the quantitation according to the present invention via the user interface 18. In this way, the present invention provides the method for executing quantitation of the protein contents based on the EMPAI and computer program for performing the above method.
- RPMI-1640 media Gibco BRL, Grand Island, NY
- 13 C 6 -LeU Carrier Isotope Laboratories, Andover, MA
- SILAC protocol by Ong et al. [4].
- Mouse neuroblastoma neuro2a cells were cultured for 13 Ce-LeU labeling in this medium.
- Whole proteins were lysed using ultrasonication with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland).
- HCT116- C9 cells were grown in a normal RPMI 1640 culture medium as described [10].
- Whole proteins were extracted with 5 mL of M-PER (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail and 5 mM dithiothreitol.
- Candidates for peptide synthesis containing at least one leucine and one tyrosine were selected considering the sequences of tryptic peptides from proteins expressed in neuro2a cells. Peptides containing methionine and tryptophane were removed to avoid the oxidation problems during sample preparation. In addition, peptides with double basic residues were removed considering the frequent missed cleavage by trypsin.
- the selected 54 peptides were synthesized using a Shimadzu PSSM-8 (Kyoto, Japan) with F-moc chemistry and were purified by preparative HPLC. Amino acid analysis, peptide mass measurement and HPLC-UV were carried out for purity and structure elucidation. Different amounts of these peptides were spiked to the peptide mixtures from neuro2a cells and purified by StageTip as described above.
- HTC-PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) mounting Valco C2 valves with 150 ⁇ m ports.
- ReproSil C18 materials (3 ⁇ m, Dr.Maisch, Ammerbuch, Germany) were packed into a self-pulled needle (100 ⁇ m ID, 6 ⁇ m opening, 150 mm length) with a nitrogen-pressurized column loader cell (Nikkyo) to prepare an analytical column needle with "stone-arch" frit [22].
- a Teflon-coated column holder (Nikkyo) was mounted on .
- Proxeon x-y-z nanospray interface (Odense, Denmark) and a Valco metal connector with magnet was used to hold the column needle and to adjust the appropriate spray position.
- the injection volume was 3 ⁇ l_ and the flowrate was 250 nL/min after a tee splitter.
- the mobile phases consisted of (A) 0.5% acetic acid and (B) 0.5% acetic acid and 80% acetonitrile.
- the three-step linear gradient of 5%B to 10% in 5 min, 10% to 30% in 60min, 30% to 100% in 5 min and 100% in 10min was employed through this study.
- Spray voltage of 2400 V was applied via the metal connector as described [22].
- MS scans were performed for 1 second to select three intense peaks and subsequent three MSMS scans were performed for 0.55 seconds each.
- An Information Dependent Acquisition (IDA) function was active for three minutes to exclude the previously scanned parent ions.
- IDA Information Dependent Acquisition
- MSMS scans 1.5 s each
- MSMS scans per one MS scan were performed.
- LCQ two MSMS scans per one MS scan were performed with AGC mode.
- the average scan cycle was 1.19 s for one MS and 1.17 s for one MSMS in average, respectively.
- the scan range was m/z 300-1400 for both QSTAR and LCQ.
- MSQuant ver1.4a was downloaded from http://msquant.sourceforge.net/, and was customized for 13 C ⁇ Leu SILAC in order to determine the ion counts in chromatograms for absolute concentration of proteins using the known amounts of the synthetic peptides.
- Protein Abundance Determination To calculate the number of observable peptides per protein, proteins were digested in silico and the obtained peptide mass was compared with the measurement scan range of mass spectrometers. In addition, the retention times under our nanoLC condition were calculated according to the procedure by Meek [23] and Sakamoto et al.[24] with our own coefficients based on approximately 3000 peptides, and peptides with too hydrophilic or hydrophobic properties were eliminated. An in-house PHP program based on the following equations (1) to (4) was written to calculate the peptide number and was used to export all data to Microsoft Excel.
- PAI is defined as
- Nobsd and Nobsbi are the number of observed peptides per protein and the number of observable peptides per protein, respectively[17]. Then, EMPAI is defined as
- Protein contents (mol%) EM pA1 x l00 ( 3 )
- MW is the molecular weight of each protein identified
- ⁇ EMPAI is the summation of EMPAI values for all identified proteins.
- HCT116-C9 cells were plated at 5.0x 10 6 cells/dish in 10-cm diameter dishes with 10 ml_ of the culture medium. After 24-h preincubation, the cells were treated for 12 h with 0.015% DMSO. Duplicate experiments were performed using Affymetrix HuGene FL arrays according to established protocols. Affymetrix GeneChip software was used to extract gene signal intensities, and two sets of data were grouped and averaged based on gene symbol.
- the Number of Identified Peptides from Single Protein with Different Concentrations Different amounts of human serum albumin (HSA) tryptic peptides were analyzed by nanoLC-ESI-MS/MS and the number of identified peptides was counted. As shown in Fig. 5A, both peak area and the number of identified peptides increased as the injection amounts increased although both curves were saturated at higher concentration of HSA. However even at the region where the peak area is linear, the number of peptides does not have linear relationship to the protein amount. Interestingly, the number of peptides shows linear relationship to logarithm of the injected amount from 3 fmol to 500 fmol (Fig. 5B). The same data was obtained from LCQ with slower scan.
- HSA human serum albumin
- T-complex protein 1 alpha subunit B 100 60867 90 6 143 0.18 0.52
- P50310 Phosphoglycerate kinase 213 44776 75 3 80 0.13 0.33
- Fig. 6A shows that there is also a linear relationship between log [protein] and the number of observed peptides normalized by the number of observable peptides per protein even when the different proteins were plotted into one graph.
- Step 1 Determination of the mass range using observed peptides and the scan range of mass spec. This step was carried out by using the actual observed mass spectrometry of the peptides, i.e., the observed peptides and the scan range of the mass spec.
- Step 2 Calculation of the predicted retention times in each observed peptides, based on Meek's equation; As explained in S122 in Fig. 4, since it is appreciated that amino acid sequences of each observed peptides can generally be determined by the MS/MS method, retention time of the observed peptides could be calculated according to the Meek's equation in which there is the relationship between the known amino acid sequence and the retention time.
- Step 3 (see S123 in Fig. 4): Determination of the retention time range using observed peptides; Similarly, this step was carried out by use of the Meek's equation.
- Step 4 (see S124 in Fig. 4): Calculation of the digested tryptic peptides without missed cleavage in silico; As described in S124 of Fig. 4, molecular weight (MW) and the predicted retention time of the digested tryptic peptides could be calculated from amino acid sequences of the digested tryptic peptides, which was determined in silico.
- Step 5 (see S125 in Fig 4): The observable peptides were sorted according to MW and the predicted retention time by use of results of S124.
- Step 6 The number of the observable peptides per protein was counted, on the basis that MW and the predicted retention time of the observable peptides fall both within the mass range (Step 121 ) and the retention time range (Step 123).
- Mass range 700-2800 Retention time range: 40-150
- EMPAI was calculated by using results of the above (3) and (4).
- the results of EMPAI are tabulated in Table 5.
- Table 5 a value of EMPAI using the sample in Table 2 was 8.345.
- PAI is really convenient to produce protein expression data from just single LCMSMS run.
- the present inventor applies this approach to compare it to gene expression in HCT116 human cancer cells.
- DNA microarray provided expression data of 4971 genes
- single LCMS run provided 402 identified proteins based on 1811 peptides with unique sequences. Bridging gene symbols with protein accession numbers resulted in total 227 genes/proteins employed for the expression comparison study. As expected, slight correlation was observed as expected from previous studies on yeast [18,26]. Interestingly, most of outliers were ribosomal proteins (see Fig. 10). It is well known that unlike prokaryotes such as E.
- EMPAI the scale for absolute protein abundance
- EMPAI is easily calculated from the output information of database search engines such as Mascot, it is possible to apply this approach to the previously measured or published dataset to add the quantitative information without any additional step.
- EMPAI can also use for relative quantitation, especially in the cases where isotope-based approaches cannot be applied because of quantitative changes that are too large for accurate measurements of ratios, because metabolic labelling is not possible or because sensitivity constraints do not allow chemical labelling techniques.
- EMPAI values of proteins in one sample can compare to those in another sample, and the outliers from the EMPAI correlation between two samples can be determined as increasing or decreasing proteins.
- This EMPAI approach can also apply to multidimensional separation-MSMS to extend the coverage of proteins. Further improvement would be possible to consider MS instrument-dependent parameters such as ionization dependence on m/z region. Since the EMPAI index can be calculated with a simple script and does not require further experimentation in protein identification experiments, we suggest its routine use in the reporting of proteomic results.
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US11/658,592 US7910372B2 (en) | 2004-07-29 | 2005-07-04 | Absolute quantitation of protein contents based on exponentially modified protein abundance index by mass spectrometry |
EP05758172A EP1787125A4 (en) | 2004-07-29 | 2005-07-04 | Absolute quantitation of protein contents based on exponentially modified protein abundance index by mass spectrometry |
JP2006554389A JP4783743B2 (en) | 2004-07-29 | 2005-07-04 | Absolute quantification of protein content based on exponentially transformed protein expression index by mass spectrometry |
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Cited By (2)
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JP2008107305A (en) * | 2006-10-27 | 2008-05-08 | Univ Of Tokyo | Protein relative quantitative determination method, its program, and its system |
WO2008143494A1 (en) * | 2007-05-22 | 2008-11-27 | Erasmus University Medical Center Rotterdam | Assay for detection of prostate cancer by means of proteolytic hsa markers |
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UA112515C2 (en) | 2009-06-03 | 2016-09-26 | Дау Аґросаєнсиз Елелсі | METHOD OF DETERMINING THE PRESENCE OF TWO OR MORE PROTEINS OF INTEREST, IN SAMPLES OF VEGETABLE ORIGIN |
EP2715367A2 (en) | 2011-06-03 | 2014-04-09 | University of South Alabama | Methods and compositions for detecting endometrial or ovarian cancer |
US9933416B1 (en) | 2013-07-30 | 2018-04-03 | Pioneer Hi-Bred International, Inc. | Detection and quantification of polypeptides in plants without a reference standard by mass spectrometry |
CN103837593B (en) * | 2014-03-18 | 2016-11-23 | 中国计量科学研究院 | Isotope dilution mass spectrometry quantitative approach after a kind of human serum protein electrophoresis |
EP3137891B1 (en) * | 2014-04-28 | 2024-01-17 | DH Technologies Development Pte. Ltd. | Multi-trace quantitation |
US9720001B2 (en) * | 2014-05-21 | 2017-08-01 | Thermo Finnigan Llc | Methods for mass spectrometric biopolymer analysis using optimized weighted oligomer scheduling |
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WO2002052259A1 (en) * | 2000-12-26 | 2002-07-04 | The Institute For Systems Biology | Rapid and quantitative proteome analysis and related methods |
WO2003054549A2 (en) * | 2001-12-08 | 2003-07-03 | Micromass Uk Limited | Method of mass spectrometry |
WO2003089937A2 (en) * | 2002-04-15 | 2003-10-30 | Thermo Finnigan, Llc | Quantitation of biological molecules |
-
2005
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- 2005-07-04 JP JP2006554389A patent/JP4783743B2/en not_active Expired - Fee Related
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WO2002052259A1 (en) * | 2000-12-26 | 2002-07-04 | The Institute For Systems Biology | Rapid and quantitative proteome analysis and related methods |
WO2003054549A2 (en) * | 2001-12-08 | 2003-07-03 | Micromass Uk Limited | Method of mass spectrometry |
WO2003089937A2 (en) * | 2002-04-15 | 2003-10-30 | Thermo Finnigan, Llc | Quantitation of biological molecules |
Non-Patent Citations (2)
Title |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008107305A (en) * | 2006-10-27 | 2008-05-08 | Univ Of Tokyo | Protein relative quantitative determination method, its program, and its system |
WO2008143494A1 (en) * | 2007-05-22 | 2008-11-27 | Erasmus University Medical Center Rotterdam | Assay for detection of prostate cancer by means of proteolytic hsa markers |
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EP1787125A1 (en) | 2007-05-23 |
US20080319676A1 (en) | 2008-12-25 |
JP4783743B2 (en) | 2011-09-28 |
EP1787125A4 (en) | 2008-10-29 |
US7910372B2 (en) | 2011-03-22 |
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