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Proteomics – The Pathway to Understanding and Decision-making in Medicine

Posted in Advanced Drug Manufacturing Technology, Clinical Diagnostics, Mass automation of plasma proteins, Pharmaceutical Discovery, Pharmaceutical Drug Discovery, Rapid automation of plasma protein pools, tagged attraction and repulsion, autosomal reactions, blood supply, catalytic reactions, cel-protein interactions, cell-cell interactions, cellular metabolism, clinical omics, covalent bonds, cytoskeleton transport, disulfide bridges, enzymes, ionic balance, isoenzymes, isoforms, LCGC?MS, macrocirculation, mass spectrometry, measurement of stochastic changes, metabolic indicators, microcirculation, mitochondria, noncovalent binding, oxidation-reduction, oxidative stress, oxyhemoglobin, pH, Protein folding, Proteomics, protin-protein interactions, tissue specificity on June 24, 2014| Leave a Comment »

Larry H Bernstein, MD, FCAP, Author and Curator

http://pharmaceuticalintelligence.com/2014/06/22/Proteomics – The Pathway to Understanding and Decision-making in Medicine

This dialogue is a series of discussions introducing several perspective on proteomics discovery, an emerging scientific enterprise in the -OMICS- family of disciplines that aim to clarify many of the challenges toward the understanding of disease and aiding in the diagnosis as well as guiding treatment decisions. Beyond that focus, it will contribute to personalized medical treatment in facilitating the identification of treatment targets for the pharmaceutical industry. Despite enormous advances in genomics research over the last two decades, there is a still a problem in reaching anticipated goals for introducing new targeted treatments that has seen repeated failures in stage III of clinical trials, and even when success has been achieved, it is temporal. The other problem has been toxicity of agents widely used in chemotherapy. Even though the genomic approach brings relieve to the issues of toxicity found in organic chemistry derivative blocking reactions, the specificity for the target cell without an effect on normal cells has been elusive.

This is not confined to cancer chemotherapy, but can also be seen in pain medication, and has been a growing problem in antimicrobial therapy. The stumbling block has been inability to manage a multiplicity of reactions that also have to be modulated in a changing environment based on 3-dimension structure of proteins, pH changes, ionic balance, micro- and macrovascular circulation, and protein-protein and protein- membrane interactions. There is reason to consider that the present problems can be overcome through a much better modification of target cellular metabolism as we peel away the confounding and blinding factors with a multivariable control of these imbalances, like removing the skin of an onion.

This is the first of a series of articles, and for convenience we shall here only emphasize the progress of application of proteomics to cardiovascular disease.

growth in funding proteomics 1990-2010

Part I.

Panomics: Decoding Biological Networks (Clinical OMICs 2014; 5)

Technological advances such as high-throughput sequencing are transforming medicine from symptom-based diagnosis and treatment to personalized medicine as scientists employ novel rapid genomic methodologies to gain a broader comprehension of disease and disease progression. As next-generation sequencing becomes more rapid, researchers are turning toward large-scale pan-omics, the collective use of all omics such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and lipoprotein proteomics, to better understand, identify, and treat complex disease.

Genomics has been a cornerstone in understanding disease, and the sequencing of the human genome has led to the identification of numerous disease biomarkers through genome-wide association studies (GWAS). It was the goal of these studies that these biomarkers would serve to predict individual disease risk, enable early detection of disease, help make treatment decisions, and identify new therapeutic targets. In reality, however, only a few have gone on to become established in clinical practice. For example in human GWAS studies for heart failure at least 35 biomarkers have been identified but only natriuretic peptides have moved into clinical practice, where they are limited primarily for use as a diagnostic tool.

Proteomics Advances Will Rival the Genetics Advances of the Last Ten Years

Seventy percent of the decisions made by physicians today are influenced by results of diagnostic tests, according to N. Leigh Anderson, founder of the Plasma Proteome Institute and CEO of SISCAPA Assay Technologies. Imagine the changes that will come about when future diagnostics tests are more accurate, more useful, more economical, and more accessible to healthcare practitioners. For Dr. Anderson, that’s the promise of proteomics, the study of the structure and function of proteins, the principal constituents of the protoplasm of all cells.

In explaining why proteomics is likely to have such a major impact, Dr. Anderson starts with a major difference between the genetic testing common today, and the proteomic testing that is fast coming on the scene. “Most genetic tests are aimed at measuring something that’s constant in a person over his or her entire lifetime. These tests provide information on the probability of something happening, and they can help us understand the basis of various diseases and their potential risks. What’s missing is, a genetic test is not going to tell you what’s happening to you right now.”

Mass Spec-Based Multiplexed Protein Biomarkers

Clinical proteomics applications rely on the translation of targeted protein quantitation technologies and methods to develop robust assays that can guide diagnostic, prognostic, and therapeutic decision-making. The development of a clinical proteomics-based test begins with the discovery of disease-relevant biomarkers, followed by validation of those biomarkers.

“In common practice, the discovery stage is performed on a MS-based platform for global unbiased sampling of the proteome, while biomarker qualification and clinical implementation generally involve the development of an antibody-based protocol, such as the commonly used enzyme linked ELISA assays,” state López et al. in Proteome Science (2012; 10: 35–45). “Although this process is potentially capable of delivering clinically important biomarkers, it is not the most efficient process as the latter is low-throughput, very costly, and time-consuming.”

Part II. Proteomics for Clinical and Research Use: Combining Protein Chips, 2D Gels and Mass Spectrometry in

The next Step: Exploring the Proteome: Translation and Beyond

N. Leigh Anderson, Ph.D., Chief Scientific Officer, Large Scale Proteomics Corporation

Three streams of technology will play major roles in quantitative (expression) proteomics over the coming decade. Two-dimensional electrophoresis and mass spectrometry represent well-established methods for, respectively, resolving and characterizing proteins, and both have now been automated to enable the high-throughput generation of data from large numbers of samples.

These methods can be powerfully applied to discover proteins of interest as diagnostics, small molecule therapeutic targets, and protein therapeutics. However, neither offers a simple, rapid, routine way to measure many proteins in common samples like blood or tissue homogenates.

Protein chips do offer this possibility, and thus complete the triumvirate of technologies that will deliver the benefits of proteomics to both research and clinical users. Integration of efforts in all three approaches are discussed, highlighting the application of the Human Protein Index® database as a source of protein leads.

leighAnderson

N. Leigh Anderson, Ph D. is Chief Scientific Officer of the Proteomics subsidiary of Large Scale Biology Corporation (LSBC).
Dr. Anderson obtained his B.A. in Physics with honors from Yale and a Ph.D. in Molecular Biology from Cambridge University
(England) where he worked with M. F. Perutz as a Churchill Fellow at the MRC Laboratory of Molecular Biology. Subsequently
he co-founded the Molecular Anatomy Program at the Argonne National Laboratory (Chicago) where his work in the development
of 2D electrophoresis and molecular database technology earned him, among other distinctions, the American Association for
Clinical Chemistry’s Young Investigator Award for 1982, the 1983 Pittsburgh Analytical Chemistry Award, 2008 AACC Outstanding
Research Award, and 2013 National Science Medal..

In 1985 Dr. Anderson co-founded LSBC in order to pursue commercial development and large scale applications of 2-D electro-
phoretic protein mapping technology. This effort has resulted in a large-scale proteomics analytical facility supporting research
work for LSBC and its pharmaceutical industry partners. Dr. Anderson’s current primary interests are in the automation of proteomics
technologies, and the expansion of LSBC’s proteomics databases describing drug effects and disease processes in vivo and in vitro.
Large Scale Biology went public in August 2000.

Part II. Plasma Proteomics: Lessons in Biomarkers and Diagnostics

Exposome Workshop
N Leigh Anderson
Washington 8 Dec 2011

QUESTIONS AND LESSONS:

CLINICAL DIAGNOSTICS AS A MODEL FOR EXPOSOME INDICATORS
TECHNOLOGY OPTIONS FOR MEASURING PROTEIN RESPONSES TO EXPOSURES
SCALE OF THE PROBLEM: EXPOSURE SIGNALS VS POPULATION NOISE

The Clinical Plasma Proteome
• Plasma and serum are the dominant non-invasive clinical sample types
– standard materials for in vitro diagnostics (IVD)
• Proteins measured in clinically-available tests in the US
– 109 proteins via FDA-cleared or approved tests
• Clinical test costs range from $9 (albumin) to $122 (Her2)
• 90% of those ever approved are still in use
– 96 additional proteins via laboratory-developed tests (not FDA
cleared or approved)
– Total 205 proteins (≅ products of 211genes, excluding Ig’s)
• Clinically applied proteins thus account for
– About 1% of the baseline human proteome (1 gene :1 protein)
– About 10% of the 2,000+ proteins observed in deep discovery
plasma proteome datasets

“New” Protein Diagnostics Are FDA-Cleared at a Rate of ~1.5/yr:
Insufficient to Meet Dx or Rx Development Needs

FDA clearance of protein diagnostics

A Major Technology Gulf Exists Between Discovery

Proteomics and Routine Diagnostic Platforms

Two Streams of Proteomics
A. Problem Technology
Basic biology: maximum proteome coverage (including PTM’s, splices) to
provide unbiased discovery of mechanistic information
• Critical: Depth and breadth
• Not critical: Cost, throughput, quant precision

B. Discovery proteomics
Specialized proteomics field,
large groups,
complex workflows and informatics

Part III. Addressing the Clinical Proteome with Mass Spectrometric Assays

N. Leigh Anderson, PhD, SISCAPA Assay Technologies, Inc.

protein changes in biological mechanisms

No Increase in FDA Cleared Protein Tests in 20 yr

“New” Protein Tests in Plasma Are FDA-Cleared at a Rate of ~1.5/yr:
Insufficient to Meet Dx or Rx Development Needs

See figure above

An Explanation: the Biomarker Pipeline is Blocked at the Verification Step

Immunoassay Weaknesses Impact Biomarker Verification

1) Specificity: what actually forms the immunoassay sandwich – or prevents its
formation – is not directly visualized

2) Cost: an assay developed to FDA approvable quality costs $2-5M per
protein

Major_Plasma_Proteins

Immunoassay vs Hybrid MS-based assays

MASS SPECTROMETRY: MRM’s provide what is missing in..IMMUNOASSAYS:

– SPECIFICITY
– INTERNAL STANDARDIZATION
– MULTIPLEXING
– RAPID CONFIGURATION PROVIDED A PROTEIN CAN ACT LIKE A SMALL
MOLECULE

MRM of Proteotypic Tryptic Peptides Provides Highly Specific Assays for Proteins > 1ug/ml in Plasma

Peptide-Level MS Provides High Structural Specificity
Multiple Reaction Monitoring (MRM) Quantitation

ADDRESSING MRM LIMITATIONS VIA SPECIFIC ENRICHMENT OF ANALYTE PEPTIDES: SISCAPA

– SENSITIVITY
– THROUGHPUT (LC-MS/MS CYCLE TIME)

SISCAPA combines best features of immuno and MS

SISCAPA Process Schematic Diagram
Stable Isotope-labeled Standards with Capture on Anti-Peptide Antibodies

An automated process for SISCAPA targeted protein quantitation utilizes high affinity capture antibodies that are immobilized on magnetic beads

Antibodies sequence specific peptide binding

SISCAP target enrichmant

Multiple reaction monitoring (MRM) quantitation

protein-quantitation-via-signature-peptides.png

First SISCAP Assay – thyroglobulin

Glycemic control in DM

Part IV. National Heart, Lung, and Blood Institute Clinical

Proteomics Working Group Report
Christopher B. Granger, MD; Jennifer E. Van Eyk, PhD; Stephen C. Mockrin, PhD;
N. Leigh Anderson, PhD; on behalf of the Working Group Members*
Circulation. 2004;109:1697-1703 doi: 10.1161/01.CIR.0000121563.47232.2A
http://circ.ahajournals.org/content/109/14/1697

Abstract—The National Heart, Lung, and Blood Institute (NHLBI) Clinical Proteomics Working Group
was charged with identifying opportunities and challenges in clinical proteomics and using these as a
basis for recommendations aimed at directly improving patient care. The group included representatives
of clinical and translational research, proteomic technologies, laboratory medicine, bioinformatics, and
2 of the NHLBI Proteomics Centers, which form part of a program focused on innovative technology development.

This report represents the results from a one-and-a-half-day meeting on May 8 and 9, 2003. For the purposes
of this report, clinical proteomics is defined as the systematic, comprehensive, large-scale identification of
protein patterns (“fingerprints”) of disease and the application of this knowledge to improve patient care
and public health through better assessment of disease susceptibility, prevention of disease, selection of
therapy for the individual, and monitoring of treatment response. (Circulation. 2004;109:1697-1703.)
Key Words: proteins diagnosis prognosis genetics plasma

Part V. Overview: The Maturing of Proteomics in Cardiovascular Research

Jennifer E. Van Eyk
Circ Res. 2011;108:490-498 doi: 10.1161/CIRCRESAHA.110.226894
http://circres.ahajournals.org/content/108/4/490

Abstract: Proteomic technologies are used to study the complexity of proteins, their roles, and biological functions.
It is based on the premise that the diversity of proteins, comprising their isoforms, and posttranslational modifications
(PTMs) underlies biology.

Based on an annotated human cardiac protein database, 62% have at least one PTM (phosphorylation currently dominating),
whereas 25% have more than one type of modification.

The field of proteomics strives to observe and quantify this protein diversity. It represents a broad group of technologies
and methods arising from analytic protein biochemistry, analytic separation, mass spectrometry, and bioinformatics.
Since the 1990s, the application of proteomic analysis has been increasingly used in cardiovascular research.

prevalence-of-cardiovascular-diseases-in-adults-by-age-and-sex-u-s-2007-2010.

Technology development and adaptation have been at the heart of this progress. Technology undergoes a maturation,

becoming routine and ultimately obsolete, being replaced by newer methods. Because of extensive methodological
improvements, many proteomic studies today observe 1000 to 5000 proteins.

Only 5 years ago, this was not feasible. Even so, there are still road blocks. Nowadays, there is a focus on obtaining
better characterization of protein isoforms and specific PTMs. Consequentl, new techniques for identification and
quantification of modified amino acid residues are required, as is the assessment of single-nucleotide polymorphisms
in addition to determination of the structural and functional consequences.

In this series, 4 articles provide concrete examples of how proteomics can be incorporated into cardiovascular
research and address specific biological questions. They also illustrate how novel discoveries can be made and
how proteomic technology has continued to evolve. (Circ Res. 2011;108:490-498.)
Key Words: proteomics technology protein isoform posttranslational modification polymorphism

Part VI. The -omics era: Proteomics and lipidomics in vascular research

Athanasios Didangelos, Christin Stegemann, Manuel Mayr∗

King’s British Heart Foundation Centre, King’s College London, UK

Atherosclerosis 2012; 221: 12– 17 http://dx.doi.org/10.1016/j.atherosclerosis.2011.09.043

a b s t r a c t

A main limitation of the current approaches to atherosclerosis research is the focus on the investigation of individual
factors, which are presumed to be involved in the pathophysiology and whose biological functions are, at least in part, understood.

These molecules are investigated extensively while others are not studied at all. In comparison to our detailed
knowledge about the role of inflammation in atherosclerosis, little is known about extracellular matrix remodelling
and the retention of individual lipid species rather than lipid classes in early and advanced atherosclerotic lesions.

The recent development of mass spectrometry-based methods and advanced analytical tools are transforming
our ability to profile extracellular proteins and lipid species in animal models and clinical specimen with the goal
of illuminating pathological processes and discovering new biomarkers.

Fig. 1. ECM in atherosclerosis. The bulk of the vascular ECM is synthesised by smooth muscle cells and composed primarily of collagens, proteoglycans and glycoproteins.During the early stages of atherosclerosis, LDL binds to the proteoglycans of the vessel wall, becomes modified, i.e. by oxidation (ox-LDL), and sustains a proinflammatory cascade that is proatherogenic

Lipidomics of atherosclerotic plaques

Fig. 2. Lipidomics of atherosclerotic plaques. Lipids were separated by ultra performance reverse phase
liquid chromatography on a Waters® ACQUITY UPLC® (HSS T3 Column, 100 mm × 2.1 mm i.d., 1.8 _m
particle size, 55 ◦C, flow rate 400 _L/min, Waters, Milford MA, USA) and analyzed on a quadrupole time-of-flight
mass spectrometer (Waters® SYNAPTTM HDMSTM system) in both positive (A) and negative ion mode (C).
In positive MS mode, lysophosphatidyl-cholines (lPCs) and lysophosphatidylethanolamines (lPEs) eluted first;
followed by phosphatidylcholines (PCs), sphingomyelin (SMs), phosphatidylethanol-amines (PEs) and cholesteryl
esters (CEs); diacylglycerols (DAGs) and triacylglycerols (TAGs) had the longest retention times. In negative MS mode,
fatty acids (FA) were followed by phosphatidyl-glycerols (PGs), phosphatidyl-inositols (PIs), phosphatidylserines (PS)
and PEs. The chromatographic peaks corresponding to the different classes were detected as retention time-mass to
charge ratio (m/z) pairs and their areas were recorded. Principal component analyses on 629 variables from triplicate
analysis (C1, 2, 3 = control 1, 2, 3; P1, 2, 3 = endarterectomy patient 1, 2, 3) demonstrated a clear separation of
atherosclerotic plaques and control radial arteries in positive (B) and negative (D) ion mode. The clustering of the
technical replicates and the central projection of the pooled sample within the scores plot confirm the reproducibility
of the analyses, and the Goodness of Fit test returned a chi-squared of 0.4 and a R-squared value of 0.6.

Challenges in mass spectrometry

Mass spectrometry is an evolving technology and the technological advances facilitate the detection and quantification
of scarce proteins. Nonetheless, the enrichment of specific subproteomes using differential solubilityor isolation of cellular
organelleswill remain important to increase coverage and, at least partially, overcome the inhomogeneity of diseased tissue,
one of the major factors affecting sample-to-sample variation.

Proteomics is also the method of choice for the identification of post-translational modifications, which play an essential
role in protein function, i.e. enzymatic activation, binding ability and formation of ECM structures. Again, efficient enrichment
is essential to increase the likelihood of identifying modified peptides in complex mixtures. Lipidomics faces similar challenges.
While the extraction of lipids is more selective, new enrichment methods are needed for scarce lipids as well as labile lipid
metabolites, that may have important bioactivity. Another pressing issue in lipidomics is data analysis, in particular the lack
of automated search engines that can analyze mass spectra obtained from instruments of different vendors. Efforts to
overcome this issue are currently underway.

Conclusions

Proteomics and lipidomics offer an unbiased platform for the investigation of ECM and lipids within atherosclerosis. In
combination, these innovative technologies will reveal key differences in proteolytic processes responsible for plaque rupture
and advance our understanding of ECM – lipoprotein interactions in atherosclerosis.

references

Virtualization in Proteomics: ‘Sakshat’ in India, at IIT Bombay(tginnovations.wordpress.com)

Proteome Portraits (the-scientist.com)

A Protease for ‘Middle-down’ Proteomics(pharmaceuticalintelligence.com)

Intrinsic Disorder in the Human Spliceosomal Proteome(ploscompbiol.org)