Jeffrey J. Porter, Justin Wildsmith, Christopher D. Melm, mark D. Schuchard, Kevin M. Ray, Dian Er Chen, Graham B.I. Scott


Introduction

Plasma Facts


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Figure 1.

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AQUA™ Depletion plasma facts


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Figure 2.Plasma Protein Depletion Workflow


Methods

High-Abundance Protein Depletion

Tryptic Digestion

LC-MS/MS Analysis

AQUA™ Analysis

An AQUA™ Peptide stock systems was ready by dissolving isotopically labeling versions that the 2 target peptides (FQNALLVR* and also GASQAGAPQGR*, respectively) in 0.1% TFA to a final concentration of 62.5 fmol/mL. Every digest sample (whole and also depleted plasma) was liquified in 20 mL that the AQUA™ Peptide share solution. Samples to be analyzed making use of an Agilent capillary 1100 HPLC coupled to a Thermo Finnigan LTQ linear ion trap mass spectrometer. Utilizing an LC-SRM method, the absolute amount of each peptide (and matching protein) was determined.

Results


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. Minimal use of this technique is allowed under a licensing arrangement with Harvard clinical School." data-cmp-is="image"/>

Figure 3. Overview the the Protein-AQUA™ Method. This method was arisen by Dr. Steve Gygi and also colleagues at Harvard medical School . Limited use of this technique is allowed under a licensing plan with Harvard clinical School.


Select an optimal tryptic peptide and also stable isotope amino acid from the succession of her protein of interest.

Order artificial AQUA™ Peptide from.

Optimize LC-MS/MS separation protocol because that quantitation.

Extract protein from biological samples and add known quantity of AQUA™ peptide.

Digest.

Analyze by LC-MS/MS or MALDI to quantitate protein of interest.

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Click on image to enlarge.




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Figure 4A) with bad resolution and also low signal-to-noise. When analyzed in depleted plasma (Figure 4C), the endogenous gelsolin peptide is easily separated indigenous interfering types and its concentration easily calculated by comparison to the AQUA™ peptide interior standard (Figures 4B and 4D)." data-cmp-is="image"/>

Conclusions


Anderson NL, Anderson NG. 2002. The human Plasma Proteome. Mol cell Proteomics. 1(11):845-867. Http://dx.doi.org/10.1074/mcp.r200007-mcp200
Gerber SA, rush J, Stemman O, Kirschner MW, Gygi SP. 2003. Absolute quantification that proteins and phosphoproteins from cell lysates through tandem MS. Proceedings the the nationwide Academy that Sciences. 100(12):6940-6945. Http://dx.doi.org/10.1073/pnas.0832254100

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