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 solution was ready by dissolve isotopically labeling versions the the two target peptides (FQNALLVR* and GASQAGAPQGR*, respectively) in 0.1% TFA to a final concentration of 62.5 fmol/mL. Every digest sample (whole and also depleted plasma) was dissolved in 20 mL that the AQUA™ Peptide stock solution. Samples to be analyzed utilizing an Agilent capillary 1100 HPLC combination to a Thermo Finnigan LTQ straight ion catch mass spectrometer. Using an LC-SRM method, the absolute quantity of every peptide (and equivalent protein) was determined.

Results


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. Minimal use the this method is permitted under a licensing arrangement with Harvard clinical School." data-cmp-is="image"/>

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


Select one optimal tryptic peptide and also stable isotope amino acid from the succession of your protein that interest.

Order fabricated 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 through LC-MS/MS or MALDI come quantitate protein the interest.

See more: What Breed Is Bolt The Superdog, Bolt (Character)


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Click on photo to enlarge.




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

Conclusions


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

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