Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Despite omics technologies being capable of generating thousands of biomarker candidates, so few actually make it to clinical use.

Many scientists point out that it’s become clear there are no simple universal strategies for the comprehensive analysis of complex proteomes. Although the application of omics technologies to biological samples generates hundreds to thousands of biomarker candidates, a small number actually make it through the pipeline to clinical use, largely due to the incredible mismatch between the large numbers of biomarker candidates and the paucity of reliable assays and methods for validation studies.

Currently, the main technology platform for systematically interrogating large numbers of proteins is based on multiple reaction monitoring (MRM) mass spectrometry (MS). However, a substantial challenge in using MRM-MS for targeted peptide analysis in clinical proteomics applications is the prevalence of interfering peptides and small molecules in the sample matrix. This problem, although well studied for small molecule analysis, is both less well recognized and far more severe for peptide analysis because peptide MRM analyses are typically carried out in an ocean of many hundreds of thousands to millions of peptides produced by digestion of the 10,000 or more proteins found in blood and other tissues.

Two approaches to using MS to find and quantitate low-abundant, potentially clinically important proteins rely on either technology tweaking by enhancing sensitivity-analyte enrichment methods that combine MS with immunity affinity, or by enriching the concentration of target analytes in complex biological samples.

Timothy D. Veenstra, Ph.D., formerly director of the laboratory of proteomics and analytical technologies at the Frederick National Laboratory for Cancer Research (formerly SAIC-Frederick), notes that the MS signal obtained from a peptide behaves differentially depending on its matrix. The environment of a given molecule affects its ionization efficiency; therefore, a solution only containing the peptide of interest will undoubtedly give a much more intense MS signal than the identical peptide within serum or plasma.

In an article in 2009 in Briefings in Functional Genomics, Dr. Veenstra illustrated this point by showing the effect of adding increasing amounts of cell lysate on the signals obtained from a heavy isotopic version of the surrogate peptide (SGGGDLTLGLEPSEEEAPR; [M + 2H]2+ m/z 962.5) for the protein HER2. The areas of the peaks, representing three transition ions that were monitored during an MRM experiment, all showed a >50% decrease when the amount of matrix added to the pure peptide was increased from 100 to 250 ng. A final addition of 1,000 ng of cell lysate to the peptide solution made each signal essentially unquantifiable.


Fundamental Need

This example, according to Dr. Veenstra and colleagues, illustrates a fundamental need for methods and strategies that enable targeting of specific proteins or peptides within complex proteomic mixtures and therefore the requirement to create a mixture that is enriched for the molecule of interest. And, he said, while simple enough in concept, in practice it is not trivial. “The previous example shows the detrimental effect that only a small amount of lysate can have on a peptide signal. Therefore, the higher the enrichment, the better the assay will be,” he wrote.

Keshishian et al., noted in Molecular Cell and Proteomics in 2007 that many protein biomarkers of clinical relevance are present at or below the nanogram/milliliter range in plasma and, therefore, inaccessible by standard MS-based methods. Using MRM coupled with stable isotope dilution MS, the investigators reported development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1–10 ng/mL range without immunoaffinity enrichment of either proteins or peptides.

Quantitative MRM assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, the investigators determined that each target protein had at least one signature peptide with a limit of quantitation in the 1–10 ng/mL range and linearity typically over two orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range.

These levels of assay performance represented, they said, up to a 1,000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS.

Writing in the Journal of Proteome Research in 2004, Anderson and Hunter noted that while direct MRM measurements in plasma digests can provide a valuable specific assay platform for biomarker validation, the addition of SISCAPA technology, (stable isotope standards and capture by antipeptide antibodies: SISCAPA) can extend MRM to lower abundance proteins by enrichment of specific target peptides. The technique, developed by N. Leigh Anderson, Ph.D., involves immobilization of antipeptide antibodies, either on magnetic beads or small columns, which are used to capture and enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the antipeptide antibody supports, electrospray or MALDI mass spectrometry is used to quantitate the peptides, both natural and labeled.

SISCAPA, its developers say, can be multiplexed, i.e., antibodies specific to a number of peptides can be added to the same sample without loss in sensitivity. Recent data suggests that up to 30 antibodies (or more) can be multiplexed. To increase throughput further, an automated workflow using 96-well plates has been demonstrated that processes up to 1,000 samples per week.

“The major challenge in really complex samples, for example human plasma, is that plasma contains all of the human proteins over a very wide range of concentrations,” Dr. Anderson told Clinical OMICs. ‘To get to a precise measurement of a specific protein you need a huge amount of cleanup—either that or you have to extensively separate the sample into all these components. In the latter case it could take a week to analyze just one sample.”


Surrogates for Proteins

SISCAPA captures peptides generated by tryptic digestion as surrogates for proteins, added Dr. Anderson.

“Using immunoaffinity enrichment, we use a specific antibody to pull the peptide out of the complicated digest. That purified peptide is what gets presented to the mass spec with little or no need for chromatography. This approach cleans up samples enough for high throughput and allows development of much more sensitive assays because you can start with a much larger sample,” he explained. “What has made this practical at this point is that the SISCAPA workflow is a completely automated process, and can be done in a push-button mode. We have been running this in 10–30 plexed assays on hundreds of samples.”

According to Dr. Anderson, the original motivation to do all this is the need to validate new clinical biomarkers. Precision and sensitivity requirements increase as you move beyond elaborate proteome discovery methods and transition to specific high-throughput assays offering clinical precision at reasonable cost. SISCAPA bridges that gap, he pointed out.

Tamburro et al., in the Journal of the American Chemical Society in 2011 explained that low abundance proteins, including biomarkers for cancer and other diseases, are invisible to mass spectrometry not only because they exist in body fluids in low concentrations, but also because they are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile.

These investigators created porous, buoyant, core-shell hydrogel nanoparticles containing novel high-affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Used for whole blood as a one-step, in-solution preprocessing technique, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa. This approach, they said, achieved a 10,000-fold effective amplification of the analyte concentration, enabling MS discovery of candidate biomarkers that were previously undetectable.

The nanoparticles also effectively protected biomarkers like interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis.

The research of Michael Super, Ph.D., at the Wyss Institute for Biologically Inspired Engineering at Harvard University, leverages protein engineering to design therapeutics and diagnostic devices to treat cancer and infectious and immunological diseases. He and his colleagues developed methods and reagents for sample extraction from complex biological environments such as human blood using broad-spectrum pathogen binding proteins attached to nanomagnetic particles. 


Sepsis Therapeutic Device

Since 2011, and with grant support from the Defense Advanced Research Projects Agency, Dr. Super and his colleagues have leveraged the Institute’s capabilities and resources to develop a sepsis therapeutic device to advance the blood-cleansing technology and help accelerate its translation to humans as a new type of sepsis therapy.

But this technology may also advance preparative technology for omics analysis by capturing and concentrating rare biomarkers from blood and other large volumes of fluids. Dr. Super and his team’s nanomagnetic bead-based technology uses a genetically engineered version of the human native mannose binding lectin (an opsonin) as a molecular hook coated onto the beads. The opsonin molecule, which occurs naturally in human blood, functions as part of the innate immune system, capturing a broad range of toxins, including over 90 different types of pathogens such as gram-negative and positive bacteria, fungi, viruses, and parasites.

Opsonin-coated beads have been used to enrich samples from individuals with sepsis for molecular and protein analysis without the need for culturing. It captures organisms circulating in the blood, thereby saving significant time and providing same-day pathogen identification.

The opsonin nanoparticles can, Dr. Super said, capture and concentrate pathogens from large volumes and provide a flexible concentration step for multiple molecular tests, including MS, PCR, and NGS, and serve as key components of a sepsis biomarker diagnostic assay.

Dr. Super explained that even the current rapid techniques for pathogen identification in sepsis are time consuming and that most patient samples are too contaminated with host proteins and DNA to use for rapid analyses. “Even the most advanced MS technology still requires you to first culture the pathogen,” he explained. “That’s a massive problem because only in about 30% of sepsis cases do you get positive cultures.”

His team’s approach is to directly concentrate the pathogens from the patient’s blood for nucleic acid sequencing.

Current mass spec algorithms were designed for MALDI-TOF MS, which depends on ribosomal proteins for characterization, according to Dr. Super. “We would have to develop a new algorithm that would use LC-MS or GCMS and look at bacterial debris, at lipids and peptides,” he continued. But, “I am still holding out hope that next-gen sequencing would work on our highly enriched bacterial populations.”

Advancing preparative techniques, these researchers believe, will make omics analysis, particularly proteomics, more accessible, rapid, and specific, getting closer to more universal strategies for analyses of complex proteomes. Proteomics continues to rapidly evolve through invention of improved sample-handling methods, use of higher-efficiency chromatography, and the introduction of faster, more sensitive and precise MS technologies.







































Patricia Fitzpatrick Dimond, Ph.D. (pdimond@genengnews.com), is technical editor at Genetic Engineering & Biotechnology News.

This article was originally published in the October 22 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to www.clinicalomics.com.

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