Proteomics/Biomarkers

Presentation

Proteomics and Drug Discovery Biomarkers List of Topics   List of Topics

 

Mass spectrometry based targeted protein quantification: methods and applications

File:Triple Q.jpg

Cross section of triple Q

Main Focus

The main focus of the paper was a review of the methods and applications of using mass spectrometry to quantify proteins, especially those that are in a concentration of less than µg/ml concentrations, in an attempt to universalize a procedure.

New terms

MALDI TOF/TOF

matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometer.

Selected reaction monitoring (SRM)

method in which a specific product ion from a specific parent ion is detected. All other ions with masses not specified in a predetermined range are filtered away leaving only ions with the mass in the range we are looking for. (source http://en.wikipedia.org/wiki/Mass_chromatogram#Selected_reaction_monitoring_.28SRM.29)

Triple quadrupole

type of MS that contains a linear series of three quadrupoles. The first and third set act as mass filters, and the second is a collision cell. This type of MS can “filter” an ion of a known mass. (source http://en.wikipedia.org/wiki/Quadrupole_mass_analyzer)

Hydrazide

class of organic compounds that share a common functional group characterized by a N-N covalent bond with one of the constituents being an acyl group. (source http://en.wikipedia.org/wiki/Hydrazide)

Biomarker

biochemical feature that can be used to measure progress of the effects of treatment or a disease. (source http://www.medterms.com/script/main/art.asp?articlekey=6685)

For this summary, we will focus on protein biomarkers. Some diseases which have protein biomarkers that show promise as a screening tool are breast cancer, Alzheimer's, leukemia, ALS, and Parkinson's . A series of six steps must be accomplished in order to successfully validate a biomarker or set of biomakers: discovery, qualification, verification, assay optimization, validation and commercialization. Once a biomarker is found and accepted, it can be used to possibly predict and prevent the disease it's related to. The summary below focuses on the quantification method of proteins in the search for and identification of protein biomarkers. By finding ways in which to universally quantify proteins, one can search for all biomarkers in one screening rather than multiple screenings, once conclusive biomarkers are identified.

Summary

With the recent breakthroughs in technology, it is conceivable that is possible to have a “universal” method or approach with minimal restrictions to quantitatively assay a wide number of proteins in search of potential biomarkers. Once a few potential biomarkers are discovered, further research can be done to confirm or refute its use in clinical applications. Another goal is to easily accumulate multiple detections in a single measurement. Measurements are taken by identifying synthetically stable isotopes attached to their respective proteins or peptides. Each isotope mimics the peptide’s endogenous counterparts allowing high selectivity.

Mass spectrometry (MS) provides us with a powerful tool to compare two different protein samples. It can be used for comparing the proteome of a diseased sample against a normal sample at a global scale. This is applied to a wide array of human diseases, with the hope that it will lead to identification of biomarkers or even pathogenesis of a disease. Traditionally, ELISA (enzyme-linked immunosorbent assay) has been the major method for the quantification of proteins with good sensitivity. Even today, it is the “gold standard” for targeted protein quantification. The major drawback with ELISA is the lack of availability of antibodies with high specificity.

First attempts to determine the amount of specific proteins were done using stable isotope dilution methods and MS approximately 20 years ago, starting with atom bombardment MS and deuterium-labeled synthetic polypeptides. Advances in MS instrumentation has increased our ability to detect candidate proteins in complicated biological samples with high sensitivity. To quantify the results, introduction of a stable isotope (containing ¹³C or ¹⁵N, for example) to selected amino acids in a reference peptide sequence provides a peptide with the same physicochemical properties, that can be readily distinguished by MS from the peptide in the target tissue or fluid. Studies have shown that full-length proteins with stable isotopes can be used in quantification of biomarkers in urine and water samples with nanomolar and picomolar level sensitivities respectively.

There is a variety of MS platforms used for quantitative proteomics, some of which are triple quadrupole (triple Q), matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF), electrospray ionization (ESI) based on QTOF MS, and an ion trap instrument using selective ion monitoring (SIM) mode. The most popular of the platforms above is the triple Q. Demonstrations have shown that it can multiplex and simultaneously target more than 50 peptides for quantification in plasma in a single measurement. For targeted quantitative analysis, coupling liquid chromatography with MALDI greatly enhances the performance of MALDI bases MS. Some advantages of this application include the ability of the techniques to be performed in parallel with each other, it can be made a highly selective, data-driven procedure, and the preservation of the sample to some degree for repeat analysis. This technique is also highlighted by its potential high throughput and excellent resolution.

One of the most important steps in quantification is sample preparation which greatly influences sensitivity. One of the most common steps used is the depletion of highly abundant proteins making it easier to enhance analytical dynamic range and the detection of proteins in low concentrations. One of the techniques performed is strong-cation exchange chromatography (SCX) which has shown to give the ability to detect peptides in the high pg/ml level, giving a 100-fold improvement over direct plasma analysis.

Post-translational modification (PTM) is an important process to understand since it is often involved in tumor progression, but can be a problem to mimic due to the complexity and structure of the sugar chains (as in glycosylation PTM). One experiment extracted N-linked glycopeptides and de-glycosylated. This resulted in the conversion of Asn to Asp and a difference in mass. This was utilized to make a synthetic polypeptide to replicate an N-linked glycopeptide in its glycosylated form.

Once of the main features of MS based quantification is for clinical applications used to identify biomarkers associated with diseases. For example, 177 protein candidates associated with stroke and cardiovascular disease in plasma have been proposed. Some biomarkers affiliated with stroke are S-100b, B-type neurotrophic growth factor, von Willebrand factor and monocyte chemotactic protein-1. Other biomarkers have been proposed to rheumatoid arthritis and breast cancer among others.

One of the main goals of the ability to quantify proteins and peptides is for personalized medicine. As technology advances, we will be able to create techniques that easily assemble multiples detection in a single measurement. Biomarkers from diseases can also be multiplexed in a single assay, allowing us to possibly diagnose multiple diseases. Ideally, a single-step preparation strategy is key, allowing for high throughput and possible an automated process, reducing the amount of human interaction and the chance of human error.

Relevance to Proteomics Course

The ability to quantify proteins using mass spectrometry is a great tool to compare a large number of proteins from a control sample with a test sample in search of biomarkers. When a noticeable difference is detected, further studies can be performed on those proteins. Major breakthroughs in MS technology give us the capability to universally approach developments to quantify a wide spectrum of proteins with little restriction. It also gives us the ability to make more detections per measurement. In the future, these approaches can give way to personal medicine giving us the ability to screen individuals by detecting multiple biomarkers from a single or multiple diseases.

References

[1] Pharmaceutical Outsourcing Decisions. SPG Media Limited. (http://www.pharmaceuticaloutsourcing.com/articles/pod003_014_power3.htm)

[2] Rifai, Nader, Gillette, Michael A., and Carr, Steven A. "Protein biomarker discovery and validation: the long and uncertain path to clinical utility". Nature Biotechnology 24, 971 - 983 (2006) (http://www.nature.com/nbt/journal/v24/n8/abs/nbt1235.html)

[3] Reynolds, Mark A., et al. "Early Biomarkers of Stroke". Clinical Chemistry 49 (2003): 1733-1739. Print. (http://www.clinchem.org/cgi/content/abstract/49/10/1733)