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 prepared by dissolve isotopically labeled versions of the two target peptides (FQNALLVR* and GASQAGAPQGR*, respectively) in 0.1% TFA come a final concentration that 62.5 fmol/mL. Each digest sample (whole and depleted plasma) was dissolved 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 straight ion catch mass spectrometer. Utilizing an LC-SRM method, the absolute quantity of every peptide (and corresponding protein) was determined.

Results


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. Minimal use of this method is allowed under a licensing plan with Harvard medical School." data-cmp-is="image"/>

Figure 3. Overview that the Protein-AQUA™ Method. This technique was emerged by Dr. Steve Gygi and colleagues in ~ Harvard medical School . Limited use the this method is permitted under a licensing arrangement with Harvard medical School.


Select one optimal tryptic peptide and also stable isotope amino mountain from the sequence of your protein the interest.

Order artificial AQUA™ Peptide from.

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

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

Digest.

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

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Click on picture to enlarge.




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Figure 4A) with bad resolution and low signal-to-noise. As soon as analyzed in depleted plasma (Figure 4C), the endogenous gelsolin peptide is quickly separated native interfering types and that is concentration readily calculated by comparison come 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 also phosphoproteins from cabinet lysates through tandem MS. Proceedings of the nationwide Academy that Sciences. 100(12):6940-6945. Http://dx.doi.org/10.1073/pnas.0832254100

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