Experts list the top challenges facing widespread adoption of proteomics in the medical laboratory industry
Year-by-year, clinical
laboratories find new ways to use mass spectrometry to
analyze clinical specimens, producing results that may be more precise than
test results produced by other methodologies. This is particularly true in the
field of proteomics.
However, though mass spectrometry is highly accurate and
fast, taking only minutes to convert a specimen into a result, it is not fully
automated and requires skilled technologists to operate the instruments.
Thus, although the science of proteomics is advancing
quickly, the average pathology laboratory isn’t likely to be using mass
spectrometry tools any time soon. Nevertheless, medical
laboratory scientists are keenly interested in adapting mass spectrometry
to medical lab test technology for a growing number of assays.
Molly Campbell, Science Writer and Editor in Genomics, Proteomics, Metabolomics, and Biopharma at Technology Networks, asked proteomics experts “what, in their opinion, are the greatest challenges currently existing in proteomics, and how can we look to overcome them?” Here’s a synopsis of their answers:
Lack of High Throughput Impacts Commercialization
Proteomics isn’t as efficient as it needs to be to be
adopted at the commercial level. It’s not as efficient as its cousin genomics. For it to become
sufficiently efficient, manufacturers must be involved.
John Yates
III, PhD, Professor, Department of Molecular Medicine at Scripps Research California
campus, told Technology
Networks, “One of the complaints from funding agencies is that you can
sequence literally thousands of genomes very quickly, but you can’t do the same
in proteomics. There’s a push to try to increase the throughput of proteomics
so that we are more compatible with genomics.”
For that to happen, Yates says manufacturers need to
continue advancing the technology. Much of the research is happening at
universities and in the academic realm. But with commercialization comes
standardization and quality control.
“It’s always exciting when you go to ASMS [the conference for the American Society
for Mass Spectrometry] to see what instruments or technologies are going to be
introduced by manufacturers,” Yates said.
There are signs that commercialization isn’t far off. SomaLogic, a privately-owned American protein
biomarker discovery and clinical diagnostics company located in Boulder, Colo.,
has reached the commercialization stage for a proteomics assay platform called SomaScan. “We’ll be
able to supplant, in some cases, expensive diagnostic modalities simply from a
blood test,” Roy
Smythe, MD, CEO of SomaLogic, told Techonomy.
The graphic above illustrates the progression mass spectrometry took during its development, starting with small proteins (left) to supramolecular complexes of intact virus particles (center) and bacteriophages (right). Because of these developments, today’s medical laboratories have more assays that utilize mass spectrometry. (Photo copyright: Technology Networks/Heck laboratory, Utrecht University, the Netherlands.)
Achieving the Necessary Technical Skillset
One of the main reasons mass spectrometry is not more widely
used is that it requires technical skill that not many professionals possess.
“For a long time, MS-based proteomic analyses were technically demanding at
various levels, including sample processing, separation science, MS and the
analysis of the spectra with respect to sequence, abundance and
modification-states of peptides and proteins and false discovery rate
(FDR) considerations,” Ruedi
Aebersold, PhD, Professor of Systems Biology at the Institute of Molecular Systems Biology (IMSB) at
ETH Zurich, told Technology
Networks.
Aebersold goes on to say that he thinks this specific
challenge is nearing resolution. He says that, by removing the problem created
by the need for technical skill, those who study proteomics will be able to
“more strongly focus on creating interesting new biological or clinical
research questions and experimental design.”
Yates agrees. In a paper titled, “Recent Technical Advances in
Proteomics,” published in F1000 Research, a peer-reviewed open research
publishing platform for scientists, scholars, and clinicians, he wrote, “Mass
spectrometry is one of the key technologies of proteomics, and over the last
decade important technical advances in mass spectrometry have driven an
increased capability of proteomic discovery. In addition, new methods to
capture important biological information have been developed to take advantage
of improving proteomic tools.”
No High-Profile Projects to Stimulate Interest
Genomics had the Human Genome Project
(HGP), which sparked public interest and attracted significant funding. One of
the big challenges facing proteomics is that there are no similarly big,
imagination-stimulating projects. The work is important and will result in
advances that will be well-received, however, the field itself is complex and difficult
to explain.
Emanuel
Petricoin, PhD, is a professor and co-director of the Center for Applied
Proteomics and Molecular Medicine at George
Mason University. He told Technology
Networks, “the field itself hasn’t yet identified or grabbed onto a
specific ‘moon-shot’ project. For example, there will be no equivalent to the
human genome project, the proteomics field just doesn’t have that.”
He added, “The equipment needs to be in the background and
what you are doing with it needs to be in the foreground, as is what happened
in the genomics space. If it’s just about the machinery, then proteomics will
always be a ‘poor step-child’ to genomics.”
Democratizing Proteomics
Alexander
Makarov, PhD, is Director of Research in Life Sciences Mass Spectrometry
(MS) at Thermo Fisher
Scientific. He told Technology
Networks that as mass spectrometry grew into the industry we have today,
“each new development required larger and larger research and development teams
to match the increasing complexity of instruments and the skyrocketing
importance of software at all levels, from firmware to application. All this
extends the cycle time of each innovation and also forces [researchers] to
concentrate on solutions that address the most pressing needs of the scientific
community.”
Makarov describes this change as “the increasing democratization of MS,” and says that it “brings with it new requirements for instruments, such as far greater robustness and ease-of-use, which need to be balanced against some aspects of performance.”
One example of the increasing democratization of MS may be
several public proteomic datasets available to scientists. In European
Pharmaceutical Review, Juan
Antonio Viscaíno, PhD, Proteomics Team Leader at the European Bioinformatics Institute (EMBL-EBI)
wrote, “These datasets are increasingly reused for multiple applications, which
contribute to improving our understanding of cell biology through proteomics
data.”
Sparse Data and Difficulty Measuring It
Evangelia
Petsalaki, PhD, Group Leader EMBL-EBI, told Technology
Networks there are two related challenges in handling proteomic data.
First, the data is “very sparse” and second “[researchers] have trouble
measuring low abundance proteins.”
Petsalaki notes, “every time we take a measurement, we
sample different parts of the proteome or phosphoproteome and
we are usually missing low abundance players that are often the most important
ones, such as transcription
factors.” She added that in her group they take steps to mitigate those
problems.
“However, with the advances in MS technologies developed by
many companies and groups around the world … and other emerging technologies
that promise to allow ‘sequencing’ proteomes, analogous to genomes … I expect
that these will not be issues for very long.”
So, what does all this mean for clinical laboratories? At the
current pace of development, its likely assays based on proteomics could become
more common in the near future. And, if throughput and commercialization ever
match that of genomics, mass spectrometry and other proteomics tools could
become a standard technology for pathology laboratories.
This new atlas of leukemia proteomes may prove useful for medical laboratories and pathologists providing diagnostic and prognostic services to physicians treating leukemia patients
Researchers at the University of Texas at San Antonio (UTSA) and the University of Texas MD Anderson Cancer Center created the online atlases—categorized into adult and pediatric datasets—to “provide quantitative, molecular hallmarks of leukemia; a broadly applicable computational approach to quantifying heterogeneity and similarity in molecular data; and a guide to new therapeutic targets for leukemias,” according to the Leukemia Atlases website.
In building the Leukemia Proteome Atlases, the researchers identified and classified protein signatures that are present when patients are diagnosed with AML. Their goal is to improve survival rates and aid scientific research for this deadly disease, as well as develop personalized, effective precision medicine treatments for patients.
To perform the study, the scientists looked at the proteomic screens of 205
biopsies of patients with AML and analyzed the genetic, epigenetic, and
environmental diversity in the cancer cells. Their analysis “revealed 154 functional
patterns based on common molecular pathways, 11 constellations of correlated
functional patterns, and 13 signatures that stratify the outcomes of patients.”
Amina Qutub, PhD, Associate Professor at UTSA and one of the authors of the research, told UTSA Today, “Acute myelogenous leukemia presents as a cancer so heterogeneous that it is often described as not one, but a collection of diseases.”
“To decipher the clues found in proteins from blood and bone marrow of leukemia patients, we developed a new computer analysis—MetaGalaxy—that identifies molecular hallmarks of leukemia,” noted Amina Qutub, PhD (above), UTSA Professor of Biomedical Engineering and one of the UTSA study’s authors. “These hallmarks are analogous to the way constellations guide navigation of the stars: they provide a map to protein changes for leukemia,” she concluded. (Photo copyright: UTSA.)
To better understand the proteomic levels associated with AML, and share their work globally with other scientists, the researchers created the Leukemia Proteome Atlases web portal. The information is displayed in an interactive format and divided into adult and pediatric databases. The atlases provide quantitative, molecular hallmarks of AML and a guide to new therapeutic targets for the disease.
The NCI predicts there will be approximately 21,540 new
cases of AML diagnosed this year. They will account for about 1.2% of all new
cancer cases. The disease will be responsible for approximately 10,920 deaths in
2019, or 1.8% of all cancer deaths. In 2016, there were an estimated 61,048
people living with AML in the US.
“Our ‘hallmark’ predictions are being experimentally tested
through drug screens and can be ‘programmed’
into cells through synthetic manipulation of proteins,” Qutub continued. “A
next step to bring this work to the clinic and impact
patient care is testing whether these signatures lead to the aggressive growth
or resistance to chemotherapy observed in
leukemia patients.
“At the same time, to rapidly accelerate research in
leukemia and advance the hunt for treatments,
we provide the hallmarks in an online compendium [LeukemiaAtlas.org] where fellow
researchers and oncologists worldwide can build from the resource, tools, and
findings.”
By mapping AML patients from the proteins present in their
blood and bone marrow, the researchers hope that healthcare professionals will
be able to better categorize patients into risk groups and improve treatment
outcomes and survival rates for this aggressive form of cancer.
The Leukemia Proteome Atlases are another example of the
trend where researchers work together to compile data from patients and share
that information with other scientists and medical professionals. Hopefully, having
this type of data readily available in a searchable database will enable
researchers—as well as clinical laboratory scientists and pathologists—to gain
a better understanding of AML and benefit cancer patients through improved
diagnosis, treatment, and monitoring.
According to the researchers, the finding could reveal athletes who removed their blood, took out the red blood cells, and transfused the cells into their bodies before competition. When conducted by medical laboratory professionals, such autologous blood therapies can enhance oxygen intake and increase performance during sports. However, these “self-transfusions” have been difficult to detect using current methods and that highlights the importance of ensuring these procedures are carried out by authorized healthcare facilities.
The World Anti-Doping Agency (WADA), an international organization aimed at research and education for doping-free sport, funded the Duke University research. WADA currently uses the Athlete Biological Passport to assess, over time, competitors’ body chemistries.
As the Duke researchers explored nucleic acids in red blood cells, they found that the cells actually do have a nucleus, contrary to popular belief. From there, they honed in on RNA.
Short RNA pieces, called microRNA (miRNA), control production of proteins in a cell, according to the researchers.
“While once thought to lack nucleic acids, red blood cells actually contain diverse and abundant RNA species,” the scientists noted in their paper. “In addition, proteomic analyses of red blood cells have identified the presence of Argonaute 2 (AGO2), supporting the regulatory function of miRNAs.”
The methodology Duke researchers followed involved these steps, among others:
Three units of blood were drawn from volunteers;
The researchers removed the white blood cells and about 80% of the plasma;
The remaining red blood cells were pure, just as they would need to be by someone doing autologous transfusion;
The researchers analyzed cell RNA samples at specific daily intervals: 1, 3, 7, 10, 14, 28, 36, and, 42 days;
They then compared samples to day 1 and recorded changes in RNA due to storage.
The researchers found:
Two types of miRNA increased during storage and two declined; and,
miR-720 had the most dramatic and consistent changes.
They concluded that finding increased miR-720 in athletes’ blood could be used as a biomarker for detecting stored red blood cells, which could indicate blood doping had taken place.
“The difficulty has been that the tests [WADA] have couldn’t tell the difference between a young blood cell and an old one,” Jen-Tsan Ashley Chi, MD, PhD, lead researcher on the study and Duke’s Associate Professor in Molecular Genetics and Microbiology, noted in the news release. “This increase in miR-720 is significant enough and consistent enough that it could be used as a biomarker for detecting stored red blood cells.” Chi is affiliated with Duke’s Center for Genomic and Computational Biology. (Photo copyright: Duke University.)
Implications for Detecting Blood Doping
How does this help clinical laboratories detect blood doping in athletes?
The researchers explained that RNA changes were, indeed, tell-tale signs of old blood cells circulating with normal cells. Those old blood cells could identify an athlete who did a self-transfusion of their blood before a competition.
However, before the test is used in sports more research is needed. Activity by the enzyme angiogenin in stored cells also is worthy of more exploration, as is its role in breaking apart larger RNA, the researchers noted.
“While autologous blood transfusions in athletes is very difficult to identify using conventional tests, it may be detectable based on the presence of red blood cells with levels of miR-720 significantly higher than the normal circulating cells. Further investigations will be necessary to identify the signals during red blood cell storage that stimulate angiogenin activation,” the study paper concluded.
Clinical Laboratories Involved in Sports Testing
In its 2017 Anti-Doping Testing Figures Report, WADA reported 322,050 samples were analyzed, a 7.1% increase from 300,565 samples in 2016. WADA accredits medical laboratories worldwide for conducting such analyses according to the organization’s code. This presents opportunities in sports medicine for medical laboratories to increase revenue through a new line of diagnostic tests.
The Duke study exemplifies how clinical laboratories can extend their services beyond patient care and enter a new realm of leveling playing fields worldwide.
Should greater attention be given to protein damage in chronic diseases such as Alzheimer’s and diabetes? One life scientist says “yes” and suggests changing how test developers view the cause of age-related and degenerative diseases
DNA and the human genome get plenty of media attention and are considered by many to be unlocking the secrets to health and long life. However, as clinical laboratory professionals know, DNA is just one component of the very complex organism that is a human being.
In fact, DNA, RNA, and proteins are all valid biomarkers for medical laboratory tests and, according to one life scientist, all three should get equal attention as to their role in curing disease and keeping people healthy.
Along with proteins and RNA, DNA is actually an “equal partner in the circle of life,” wrote David Grainger, PhD, CEO of Methuselah Health, in a Forbes opinion piece about what he calls the “cult of DNA-centricity” and its relative limitations.
Effects of Protein Damage
“Aging and age-related degenerative diseases are caused by protein damage rather than by DNA damage,” explained Grainger, a Life Scientist who studies the role proteins play in aging and disease. “DNA, like data, cannot by itself do anything. The data on your computer is powerless without apps to interpret it, screens and speakers to communicate it, keyboards and touchscreens to interact with it.”
“Similarly,” he continued, “the DNA sequence information (although it resides in a physical object—the DNA molecule—just as computer data resides on a hard disk) is powerless and ethereal until it is translated into proteins that can perform functions,” he points out.
According to Grainger, diseases such as cystic fibrosis and Duchenne Muscular Dystrophy may be associated with genetic mutation. However, other diseases take a different course and are more likely to develop due to protein damage, which he contends may strengthen in time, causing changes in cells or tissues and, eventually, age-related diseases.
“Alzheimer’s disease, diabetes, or autoimmunity often take decades to develop (even though your genome sequence has been the same since the day you were conceived); the insidious accumulation of the damaged protein may be very slow indeed,” he penned.
“But so strong is the cult of DNA-centricity that most scientists seem unwilling to challenge the fundamental assumption that the cause of late-onset diseases must lie somewhere in the genome,” Grainger concludes.
Shifting Focus from Genetics to Proteins
Besides being CEO of Methuselah Health, Grainger also is Co-Founder and Chief Scientific Advisor at Medicxi, a life sciences investment firm that backed Methuselah Health with $5 million in venture capital funding for research into disease treatments that focus on proteins in aging, reported Fierce CEO.
Methuselah Health, founded in 2015 in Cambridge, UK, with offices in the US, is reportedly using post-translational modifications for analysis of many different proteins.
“At Methuselah Health, we have shifted focus from the genetics—which tells you in an ideal world how your body would function—to the now: this is how your body functions now and this is what is going wrong with it. And that answer lies in the proteins,” stated Dr. David Grainger (above), CEO of Methuselah Health, in an interview with the UK’s New NHS Alliance. Click on this link to watch the full interview. [Photo and caption copyright: New NHS Alliance.]
How Does it Work?
This is how Methuselah Health analyzes damaged proteins using mass spectrometry, according to David Mosedale, PhD, Methuselah Health’s Chief Technology Officer, in the New NHS Alliance story:
Protein samples from healthy individuals and people with diseases are used;
Proteins from the samples are sliced into protein blocks and fed slowly into a mass spectrometer, which accurately weighs them;
Scientists observe damage to individual blocks of proteins;
Taking those blocks, proteins are reconstructed to ascertain which proteins have been damaged;
Information is leveraged for discovery of drugs to target diseases.
Mass spectrometry is a powerful approach to protein sample identification, according to News-Medical.Net. It enables analysis of protein specificity and background contaminants. Interactions among proteins—with RNA or DNA—also are possible with mass spectrometry.
Methuselah Health’s scientists are particularly interested in the damaged proteins that have been around a while, which they call hyper-stable danger variants (HSDVs) and consider to be the foundation for development of age-related diseases, Grainger told WuXi AppTec.
“By applying the Methuselah platform, we can see the HSDVs and so understand which pathways we need to target to prevent disease,” he explained.
For clinical laboratories, pathologists, and their patients, work by Methuselah Health could accelerate the development of personalized medicine treatments for debilitating chronic diseases. Furthermore, it may compel more people to think of DNA as one of several components interacting that make up human bodies and not as the only game in diagnostics.
At least that’s what researchers at ETH Zurich (ETH), an international university for technology and natural sciences, have concluded. They published the results of their study in Cell.
“Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environments,” the researchers wrote. “Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.”
Interactomics address interactions between proteins and small molecules, according to an article published in Technology Networks. The terms “interactomics” and “omics” were inspired by research that described, for the first time, the interactions and relationships of all proteins and metabolites (A.K.A, small molecules) in the whole proteome.
Medical laboratories and anatomic pathologists have long understood the interactions among proteins, or between proteins and DNA or RNA. However, metabolite interactions with packages of proteins are not as well known.
These new omics could eventually be an important source of diagnostic biomarkers. They may, one day, contribute to lower cost clinical laboratory testing for some diseases, as well.
Metabolite-Protein Interactions are Key to Cellular Processes
The ETH researchers were motivated to explore the interplay between small molecules and proteins because they have important responsibilities in the body. These cellular processes include:
“Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells. But our knowledge of the metabolite-protein interaction lags behind our understanding of protein-protein or protein-DNA interactomes,” the researchers wrote in Cell.
Leveraging Limited Proteolysis and Mass Spectrometry
The researchers used limited proteolysis (LiP) technology with mass spectrometry to discover metabolite-protein interactions. Results aside, experts pointed out that the LiP technology itself is significant.
“It is one of the few methods that enables the unbiased and proteome-wide profiling of protein conformational changes resulting from interaction of proteins with compounds,” stated a Biognosys blog post.
Biognosys, a proteomics company founded in 2008, was originally part of a lab at ETH Zurich.
The ETH team focused on the E. coli bacterial cell in particular and how its proteins and enzymes interact with metabolites.
“Although the metabolism of E. coli and associated molecules is already very well known, we succeeded in discovering many new interactions and the corresponding binding sites,” Paola Picotti, PhD, Professor of Molecular Systems Biology at ETH Zurich, who led the research, told Technology Networks. “The data that we produce with this technique will help to identify new regulatory mechanisms, unknown enzymes and new metabolic reactions in the cell,” she concluded. (Photo copyright: ETH Zurich.)
More than 1,000 New Interactions Discovered
The study progressed as follows, according to Technology Networks’ report:
“Cellular fluid, containing proteins, was extracted from bacterial cells;
“Strikingly, more than 80% of the reported interactions were novel and about one quarter of the measured proteome interacted with at least one of the 20 tested metabolites. This indicates that the metabolite-protein interaction network is vast and largely uncharted,” Papp stated in an ETH Zurich Faculty of 1000 online article.
According to Technology Networks, “Picotti has already patented the method. The ETH spin-off Biognosys is the exclusive license holder and is now using the method to test various drugs on behalf of pharmaceutical companies.”
The pharmaceutical industry is reportedly interested in the approach as a way to ascertain drug interactions with cellular proteins and their effectiveness in patient care.
The ETH Zurich study is compelling, especially as personalized medicine takes hold and more medical laboratories and anatomic pathology groups add molecular diagnostics to their capabilities.
