Quantitation of biological molecules

A technology for biological samples and mixtures, which is applied in the field of analysis technology for peptide identification and quantification, and can solve problems such as expensive isotope labeling reagents

Inactive Publication Date: 2005-11-02
THERMO FINNIGAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These techniques obviate the need for 2D GE and densitometry, but create an entirely different set of problems
May be difficult to achieve with rare stable isotopes (such as 18 0) Complete replacement of natural isotopes (such as 16 0) to generate a sta...

Method used

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  • Quantitation of biological molecules
  • Quantitation of biological molecules
  • Quantitation of biological molecules

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0058] The disclosed method was used for a mixture of five standard proteins - bovine albumin, equine hemoglobin, equine ferritin, equine cytochrome, and equine myoglobin. The first four proteins were maintained at a constant concentration (200 fmol), while the concentration of the fifth protein (myoglobin) varied widely. The peak area of ​​the protein digest was normalized to that of the albumin digest. The whole process was repeated three times. The RSD was 20% after three measurements and the calculated peak areas were constant for the four constant concentration protein digests. The relative peak area of ​​the fifth protein (myoglobin) showed a linear increase with increasing concentration from 10 fmol to 1000 fmol.

[0059] Sample Preparation

[0060] Five proteins were purchased from Sigma (St. Louis, MO) as lyophilized powders: bovine albumin, A-7638; horse hemoglobin, H-4632; horse ferritin, A-3641; horse myoglobin, M- 0630; Equine Cytochrome C, C-7752. Solvents a...

Embodiment 2

[0075] Lyophilized protein samples (1 mg human serum and 1 mg horse myoglobin, Sigma-Aldrich, St. Louis, Missouri, USA) were dissolved in 1 ml ammonium bicarbonate buffer (100 mM pH 8.5) and 3 μl DTT (1M, Sigma-Aldrich , St.Louis, Missouri, USA). The mixture was incubated at 37°C for 30 minutes. To alkylate the protein, 7 μl of iodoacetic acid (1M in 1M KOH, Sigma-Aldrich, St. Louis, MO, USA) was added and the mixture was incubated for an additional 30 minutes at room temperature in the dark. 13 [mu]l DTT (1M) was added to quench iodoacetic acid. 20 μl of trypsin (0.5 mg / ml, Promega, Madison, Wisconsin, USA) was added to digest the reduced and alkylated protein. The mixture was incubated at 37°C for 6 hours, then an additional 20 µl of trypsin (0.5 mg / ml) was added and the incubation was continued at 37°C for 16 hours.

[0076] Aliquots of sample digests (as indicated below) were placed in wells of a 96-well plate. Plates were sealed with plastic film to reduce evaporation...

Embodiment 3

[0084] Eleven aliquots containing different amounts (ranging from 10 fmol to 100 pmol) of myoglobin digest were analyzed by LC / MS / MS and the peak areas of five selected peptides were calculated. Experiments were repeated three times to ensure reproducibility. The peak area increases with increasing peptide concentration injected. In this experiment, the lower limit of peak detection was 10 fmol and the upper limit was 100 pmol. The peak areas for all five myoglobin peptides were combined and plotted against the amount of myoglobin. From 10fmol to 100pmol, the peak area is linearly related to the myoglobin concentration (r 2 =0.991), and the results were reproducible. A summary of the results is shown in Table 4 and Figure 11 . It should be noted that peak areas with a value of 0 (see Table 4) cannot be displayed on the logarithmic scale, but are included in the linear regression.

[0085] concentration

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Abstract

Methods and apparatus, including computer program products, for quantifying peptides in a peptide mixture. A peptide mixture containing a plurality of peptides is received. One or more peptides are separated from the peptide mixture over a period of time. One or more of the peptides separated at a particular time are subjected to mass-to-charge analysis and an abundance of one or more of the mass analyzed peptides is calculated. A relative quantity for the one or more mass analyzed peptides is calculated by comparing the calculated abundance of the peptides with an abundance of one or more peptides in a reference sample that is external to the first peptide mixture. The techniques can be applied to arbitrary peptides, without requiring the use of differential mass labeling, and can be applied to other biological molecules, such as nucleic acids and small molecules.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 60 / 373,007, filed April 15, 2002, which is incorporated herein by reference. technical field [0003] This application relates to analytical techniques for the identification and quantification of polypeptides. Background of the invention [0004] Two-dimensional gel electrophoresis (2D GE) has been the standard method for the separation and quantification of protein mixtures for many years. Binding proteins to different dyes (staining), such as Coomassie blue, or using radioactive labels such as 32 P, making it possible to visualize protein spots on the gel. After scanning the gel, the "darkness" of the spot is measured using densitometry and quantitative information is obtained. In the 1990s, mass spectrometry (MS) became a popular tool for identifying proteins after in-gel digestion....

Claims

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Application Information

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IPC IPC(8): G01N27/62C07K1/16G01N30/72G01N30/86G01N30/88G01N33/68
CPCG01N33/6848G01N33/6842
Inventor P·V·邦达尔科T·A·沙尔特D·H·彻里尤斯
Owner THERMO FINNIGAN
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