# Proteomics/Biomarkers

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### Mass spectrometry based targeted protein quantification: methods and applications

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### 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.

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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>)

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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>)

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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>)

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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 [^1]. 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[^2].
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 ^13^C or ^15^N, 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[^3]. 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)


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