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Emerging Proteomic Technologies: MALDI-TOF for Clinical Protein Quantitation

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This story is part of an ongoing series on emerging proteomic technologies. See here for the complete list of stories in the series.

NEW YORK (GenomeWeb) – Asked recently by GenomeWeb what he saw as one of the most interesting emerging proteomic technologies, SISCAPA Assay Technology CEO Leigh Anderson highlighted TOF-based quantitation, in particular MALDI-TOF-based quantitation.

In one sense, it's a curious choice, as Anderson has over the last decade been a key figure in pushing triple quad-based multiple-reaction monitoring protein assays toward clinical viability. He continues to do so via his work at SAT, which applies its technology to, among other things, the development of SISCAPA assays that can replace problematic immunoassays currently in clinical use. The assays use antibody-based peptide enrichment prior to MRM analysis to improve sensitivity and throughput. That said, Anderson has more recently begun exploring the use of MALDI-TOF mass spec for targeted protein quantitation, finding that the approach has certain potential advantages over triple quad-based MRM-MS.

MRM-MS is generally considered the most likely route through which proteomic assays will move to the clinic, and recent years have seen the launch of several such assays, including Integrated Diagnostic's Xpresys Lung test for lung cancer, Sera Prognostics' PreTRM test for risk of preterm birth, and thyroglobulin tests from a variety of clinical laboratories.

MALDI has also had some clinical success, most notably Biodesix's Veristrat test and in the field of clinical microbiology, where Bruker's MALDI Biotyper and BioMériuex's Vitek MS systems are, arguably, the most widely adopted mass spec-based clinical proteomics technologies to date.

Nonetheless, Anderson noted, "the analytical world currently assumes that triple quads offer the best quantitation."

However, he added, TOF analyzers, including MALDI-TOF instruments, "probably allow better precision in heavy-light ratio measurements," such as peptide quantitation assays using labeled internal standards.

This is because TOFs measure both the heavy and light ions in the same spectrum, meaning  that "these populations of molecules are entering the mass spec at the same instant," Anderson said.

On the other hand, "MRMs, for all their power, are always measuring the heavy and light peptides at different times, so any variation in the electrospray source will cause noise in the ratio," he said, adding that his lab has managed to achieve coefficients of variation with MALDI-TOF as low as 1 to 2 percent, better than the 2.5 to 5 percent they are currently able to achieve using a triple quad.

A review published by researchers from the University of Colorado, Arizona State University, MALDI-TOF vendor SimulTOF Systems, and others in the current issue of Clinical Chemistry, looking at the use of MALDI-MS in the clinic, observed that MALDI's poor reputation as a quantitative technology stems in part from the limitations of early instruments, specifically their relatively slow lasers which meant that typically, only a small fraction of the MALDI matrix containing the target sample was analyzed. This led to poor reproducibility across runs and samples, making these instruments poor choices for quantitative work.

More recently, however, advances such as faster lasers and improved matrices have significantly upped the reproducibility of MALDI-based protein quantitation. For instance, the Clinical Chemistry authors noted, early MALDI systems operated at laser pulse rates in the range of 10 Hz. By way of comparison, Bruker's recently released UltraFlextreme MALDI-TOF/TOF operates at speeds as fast as 2 kHz, and SimulTOF System's SimulTOF 300 instrument reaches speeds up to 5 kHz.

Technologies enabling researchers to create smaller MALDI spots could also help increase assay precision and sensitivity. Sample prep firm Labcyte, for example, has been collaborating with Stanford University researcher Mark Stolowitz on using the company's acoustic liquid handling system for spotting samples in MALDI assays.

Stolowitz is also planning a project in which he and researchers at the Fred Hutchinson Cancer Research Center will compare the performance of MALDI and triple quad-based MRM protein quantitation assays, an effort he told GenomeWeb will likely begin in April.

Beyond their ability to perform highly precise protein quantitation, modern MALDI mass specs offer certain other advantages compared to triple quads that make them an attractive clinical platform. Among the most notable is the fact that they don't require upfront LC separation, which allows for high throughput and relative simplicity of operation.

"Anything that gets rid of the LC separation improves the robustness of MS immensely," Anderson said. "For this reason, I think there is a good case that non-LC systems like MALDI, when combined with a specific enrichment technique [like antibody-based enrichment] that simplifies the sample enough to eliminate the need for LC separation, will emerge as the best clinical MS platforms."

Indeed, in a separate article in the current Clinical Chemistry, Lorin Bachmann, co-director of clinical chemistry at the Virginia Commonwealth University Health System, cited the need for LC separation as a major issue facing broad clinical adoption of mass spec.

In the first place, she noted, the complexity of LC systems presents a challenge for clinical operators.

"The quality of the LC data depends on technical factors such as how evenly the LC tubing is cut, how precisely the frits are set, and how carefully system pressures are maintained," Bachmann said. "Developing proficiency for these sorts of tasks requires intensive training and highly committed personnel. The complexity of current-generation LC systems has created a barrier to adoption of MS assays in our laboratory, which is resource limited and unable to hire personnel with expertise to maintain the systems."

Additionally, she said, "LC still remains the major rate-limiting step to throughput. MS systems will likely not be able to compete with standard immunoassay analyzers in terms of throughput and turnaround time until analyses can be performed in a nearly concurrent manner without substantially increasing the number of system failure points."

As Anderson suggested, MALDI offers a potential solution to this problem because samples can be prepared in batches and run without upfront LC, allowing such systems to process hundreds or even thousands of samples per day. For instance, in 2014 a team led by researchers from ASU and Thermo Fisher Scientific published a study in PLOS One detailing a MALDI-based workflow capable of quantifying human insulin-like growth factor 1 levels with throughput exceeding 1,000 samples per day.

With such advantages in mind, several groups are developing MALDI-based workflows for protein quantitation, for instance Anderson and his collaborators; Stanford's Stolowitz; and ASU researchers, including Dobrin Nedelkov and Randall Nelson.

Scientists at University of Victoria spinout MRM Proteomics are also looking at MALDI's clinical proteomics potential. CEO Gary Kruppa, Bruker's former vice president for business development, highlighted in a 2014 interview with GenomeWeb the possibility of developing clinical protein quantitation assays for Bruker's MALDI Biotyper instrument, which has received a variety of regulatory clearances, including US Food and Drug Administration 510(k) clearance, for clinical microbiology work.

In 2013, Bruker and MRM Proteomics launched a collaboration, using MRM's immune-MALDI (iMALDI) workflow to develop high-throughput immuno-MALDI (iMALDI) mass spec-based assays for determining genetic hemoglobin variants and diabetes risk for use on the MALDI Biotyper platform.

MRM is also interested in developing protein-based microbiology assays complementing the Biotyper's microbial ID capabilities, "things like markers for sepsis, markers for viral versus bacterial infection," Kruppa said. "There are a lot of things outside [bacterial] ID that could be done by iMALDI."

The company is also developing MALDI assays for plasma protein markers outside of microbiology, including an iMALDI assay for angiotensin I, which is used as a measure of plasma renin activity, a marker of primary aldosteronism.

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