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SciClips Open Innovation (Proteomics/Biomarkers/Metabolomics) Vol.1 Page 2B (2009)

A Universal Immuno-MS Method for the Absolute Quantitation of Proteins and Peptides

Contributor: Proteora
Published online on: Feb 09 2009

Concept: Use a chemical and photo cleavable peptides or a chemical molecule attached to a ligand to quantitate an analyte from a complex mixture or purified samples. The peptides can be unmodified or modified with isotopes or other chemical ligands. The ligand can be antibodies, proteins, peptides, DNA or small molecules such biotin etc.

Background: The absolute quantitation of peptides or proteins has been reported using mass spectrometric approaches. These techniques include the use of isotopically labeled peptides as standards (AQUA), immuno-MS approaches and other mass spectrometric methods (1-5). In all these methods, except the immuno-MS approach, labeled peptides are mixed with a complex mixture for quantitating the peptide or protein of interest. In case of immuno-MS approaches, specific proteins or peptides are captured first and then quantiate by mass spectrometry. Isotopically labeled peptides or proteins are used as controls in these experiments. Here I propose a universal method for the quantitation of proteins using a very novel approach. This approach can be used for the quantitation of other biomolecules such as DNA, lipids and small molecules (for metabolomics studies).

1. Isotopic labeling and digestion of proteins are not required. This can reduce variations that can occur from protease digestion and multiple sample handling steps such as HPLC fractionation etc.
2. A single reagent can be used for the quantitation of any proteins or peptides. There is no need to make multiple peptides from multiple proteins like other quantitation methods.


The possible advantages of this method are:

A. Absolute quantitation of biomarker proteins (sandwich-ELIS approach)

The idea is to use attach a peptide sequence of known mass, ?signature peptide?, to antibody and use this antibody to quantitate a specific protein/peptide in the test sample. The ?signature peptide? should have a photocleavable or chemical cleavable site so that the ?signature peptide? attached to antibodies can be cleaved off and quantitate using mass spectrometry. The other properties of the ?signature peptide? are i) should be easily ionizable or ii) can be isotopically labeled. The ?signature peptide? can be attached to antibodies using amine chemistry, similar to the methods used for attaching enzymes (HRS or AP) or small molecules such as biotin to antibodies. If we attach the ?signature peptide? to a secondary antibody, we can use this antibody for quantitating multiple proteins, without the need of modifying individual antibodies. The absolute qunatitation peptides can be derived from the experiments using known amount of control purified biomarker proteins. Described below is a method using a secondary antibody approach (a schematic representation of this concept is shown in Fig. 1):



1. Immobilize a primary antibody (monoclonal or polyclonal) to a test protein ( e.g. biomarker protein such as PSA) in a sold phase such as ELISA plates. Block the unbound surface with BSA or milk proteins. To one set of ELISA plates titrate known concentrations of purified biomarker protein. This will generate a standard curve for the quantitation of proteins. To another set of plates add the test sample such as serum, tissue extracts etc.
2. Incubate for a while and wash the plates (3-4 times)
3. Add another primary antibody to the test proteins. This antibody should be different from the first immobilized antibody. In other words, if you use a mouse primary antibody, you have to antibodies that are generated in other organisms other than mouse.
4. Incubate for a while, wash couple of times.
5. Add the secondary antibody labeled with ?signature peptide? to the mixture. Please remember that the secondary antibody should be against the second primary antibody. For example, if you used a primary antibody raised in rabbit (ant-rabbit), your secondary antibody should be goat anti-rabbit or horse anti-rabbit antibodies.
6. Incubate for a while, wash couple times.
7. Incubate washed plates in respective buffer for the elution. If you use photocleavable ?signature peptide?, add respective buffers and right conditions for the peptide cleavage. If the peptide contains an acid cleavable linker, incubate the plates in acid conditions. Please remember that if you use acidic conditions, antibodies will also be removed. The eluted sample will contain antibodies, proteins and peptides, which may not affect the down steam mass spectrometry analysis.
8. Quantitate the amount of peptides using mass spectrometry and calculate the concentration of peptides from the standard curve you have generated.


For the quantitation using the above approach, you have to do two separate MS analysis; one for the control and the other for the test sample. We can combine these two analysis in one experiment. This can be achieved by using two ?signature peptides? that are slightly different in molecular mass. For example, we will use one ?signature peptide? for the control sample and another ?signature peptide? for the test sample. The two ?signature peptides? will be eluted and analyzed in a single MS analysis. A schematic representation of this technique is shown in Fig. 2.



The above approach is based on sandwich ELISA and one could label the primary antibody instead of secondary antibodies. This approach would eliminate the use of secondary antibody. However, if you make a secondary antibody labeled with the ?signature peptide?, you can use this antibody for the detection of multiple proteins/peptides.



B. Absolute quantitation of proteins in solution, fixed cells tissues and tissue mciroarrays using single primary antibodies

The absolute quantitation of peptides or proteins has been reported using mass spectrometric approaches. These techniques include the use of isotopically labeled peptides as standards (AQUA), immuno-MS approaches and other mass spectrometric methods (1-5). In all these methods, except the immuno-MS approach, labeled peptides are mixed with a complex mixture for quantitating the peptide or protein of interest. In case of immuno-MS approaches, specific proteins or peptides are captured first and then quantiate by mass spectrometry. Isotopically labeled peptides or proteins are used as controls in these experiments. Here I propose a universal method for the quantitation of proteins using a very novel approach. This approach can be used for the quantitation of other biomolecules such as DNA, lipids and small molecules (for metabolomics studies).


The quantitation method proposed here can be used for absolute quantitation of isolated biomarker proteins or protein in fixed cells or tissues (including microarrays). In this approach a primary antibody labeled with a ?signature peptide? will be used. In this approach, primary or secondary antibodies can be used for ?signature peptides? labeling.

i) Quantitation proteins using nitrocellulose sheets or nitrocellulose coated glass slides We can develop a method for the absolute quantitation of proteins without using two primary antibodies (sandwich-ELISA approach) as explained earlier. In this approach, the proteins will be immobilized to membrane such as nitrocellulose and quantitate the amount of protein expressed using. The control and test proteins will be analyzed using antibodies with two ??signature peptide? labeled antibodies. We can spot the protein mixture or cell/tissue extracts directly onto a nitrocellulose membrane and quantitate the protein of interest using primary or secondary antibodies labeled with ?signature peptides?. The control and test proteins will be analyzed using antibodies with two ??signature peptides? A schematic representation of this idea is shown in Fig.3.

ii) Quantitation protein using fixed cells/tisseus or tissue microarray slides The same approach proposed for nitrocellulose membrane can be used for the quantitation of proteins in fixed cells or tissue sections or in tissue microarrays. The primary or secondary antibodies labeled with ?signature peptides? will be used in these experiments.

iii) In-gel quantitation of proteins from 1D and 2D gels The same approach proposed for nitrocellulose membrane can be used for the quantitation of proteins from 1D or 2 D gels. The primary or secondary antibodies labeled with ?signature peptides? will be used in this approach. The gel slices can be cut; the peptides will be eluted and analyzed by MS. This approach can be used for in-gel based quantitation of post-translationally modified proteins



C. Absolute quantitation of glycosylated marker proteins

The glycosylated proteins have been identified as potential biomarkers for various diseases (6-8). We have proposed a method to quantitate a glycosylated marker protein using a single antibody. This approach can be used for studying relative quantitation of glycosylated proteins. The idea is to attach a ?signature peptide? to lectin and use this labeled lectin to quantitate antibody captured glycoprotein/s. The proposed methodology is explained below. A schematic representation of this technology is shown in Fig. 4

1. Attach the signature peptide to a lectin such wheat germ agglutinin Concavalin-A etc. Amine based chemistry can be used for the attachment of signature peptide to lectins.
2. Immobilze the antibody of protein of interest (glycosylated protein e.g. HCG). Please note that the carbohydrate chain in the antibody has to modified or eleminated. This can be done by the method developed described very recently (Ref). We can also modify the carbohydrates by adding unlabeled lectins to the immobilized antibodies. The carbohydrate chains in the antibodies has to be blocked to prevent the binding of labeled lectins to antibodies rather than to the captured glycoproteins.
3. Add the cell lysate, serum and capture the protein from the test sample.
4. Then add the peptide labeled lectin
5. Incubate washed plates in respective buffer for the elution.
6. Quantitate the amount of peptides using mass spectrometry and calculate the concentration of peptides form the standard curve you have generated.

Note: We can also use the nitrocellulose membrane based approach for the quantitation of glycoproteins as explained in section B. The ?signature peptide? labeled lectins (similar to lectin blotting (9)) will be used in this approach. In-gel based approaches can also be used (as explained earlier).





D. Absolute quantitation of phosphorylated marker proteins

The idea is capture the protein using protein specific antibody and then quantitate the phosphorylation using phospho amino acid specific antibodies (labeled with ?signature peptides?). This approach is similar to method proposed using sandwich-ELSIA (section A and B).
Following is the proposed method.
1. Attach the signature peptide to a secondary antibody or to a primary antibody.Amine based chemistry can be used for the attachment of signature peptide to antibodies.
2. Immobilize one set of primary antibody to the protein of interest
3. Add the cell lysate, serum and capture the protein from the test sample.
4. Then add a primary antibody specific to phosphorylated amino acid (such as phosphor serine/threonine/tyrosine or a phosphorylated domain specific antibody)
5. Incubate and wash the plates in respective buffesr for the elution.
6. Quantitate the amount of peptides using mass spectrometry and calculate the concentration of peptides form the standard curve generated using purified proteins.



E. Absolute quantitation of other post-translationally modified marker proteins

The above mentioned antibody based methods can be sued for the abosolute quantitation of other post-translationally modified marker proteins. These modifications include, ubquitinylation, acetylation, nitrosyaltion etc.

E. Absolute qunatitation of modified vs. modified proteins in a sample

Most often, it is necessary to determine to quantitate the concentration of a particular modified and unmodified proteins present in the sample. The best example is the quantitation of glycosylated human condotrpin hormone (HCG) and non-glycosylated HCG. We can use a very simple assay for the absolute or relative quantitation of glycosylated vs. non-glycosylated HCG in a test sample.. A schematic representation of this idea is shown Fig. 5. In this idea two types or marker peptides will be used simultaneously.
This is how this idea works (e.g. HCG):
Following reagents are needed:
a. HCG primary antibody (preferably a monlclonal antibody that reacts with a single epitope) labeled with one type of photocleavable marker peptide
b. A lectin labeled with second photocleavable marker peptide. This peptide will have different molecular weight or may be isotopically labeled for minor molecular weight differences.
c. Immobilize the antibody to HCG (non-glycosylated form) onto an ELISA plate or beads (magnetic or non-magnetic)
d. Add the serum or other forms of test sample to the immobilized HCG antibody plates or beads
e. Wash the plates and add the photocleavable peptide labeled antibody and the lectin. Incubate for while
f. Wash, and elute the peptide and analyze by mass spectrometry.
g. This will give the absolute or relative quantitation of glycosylated vs non-glycosylated forms of HCG. The absolute quantitation can be done by using a standard curve prepared using purified proteins.




Other applications of this technology:

1. The above mentioned idea can be used in conjunction with bead based approaches
2. The technology can be used for the quantitation of post-tranlationally modified proteins/peptides such a phosphorylation, , acetylation etc
3. It can be used for the quantitation of small molecules such a metabolites
4. For the quantitation of cell surface proteins
5. For protein-protein interaction studies (labeling the partner protein with the signature peptide)


References

1.Molecular & Cellular Proteomics 6:2139-2149, 2007
2. Molecular & Cellular Proteomics 5:573-588, 2006
3.Molecular & Cellular Proteomics 6:2212-2229, 2007
4. US Patent Application#20080176333
5. PNAS 100: 12 6940-6945, 2003
6. Journal of Cellular Biochemistry 104:136 ? 149,2007
7. J Am Soc Nephrol 15:1677-1689, 2004
8. Clinical Proteomics 4: 114-24 (2008)
9. Trends Glycoscience Glycotechnology 16: S6, 2004



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