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SISCAPA-Based Biomarker Quantitation Moving to MALDI MS in Pursuit of Higher Throughput, Lower Costs


By Adam Bonislawski

Pursuing lower costs and higher throughput, several proteomics researchers are investigating the use of MALDI mass spectrometry for SISCAPA assays.

The SISCAPA, or stable isotope standards and capture by anti-peptide antibodies, workflow, which combines antibody-based peptide enrichment with mass spec to increase the sensitivity and specificity of mass spec instruments, has typically been coupled to selected-reaction monitoring assays on triple quadrupole instruments. However, advances in sample prep procedures and the use of high-quality antibodies have opened up the possibility of moving the assays to a MALDI-TOF platform, which could enable faster, simpler, and lower-cost analyses.

The challenge “is to develop the most efficient, cost-effective system for doing these [SISCAPA] assays in high throughput for both research and clinical [use],” said Leigh Anderson, developer of the technology. “And clearly MALDI has the potential to do really high-throughput, inexpensive analytical work.”

Triple quadrupole-based SRM analysis has been the “default gold standard” for protein quantitation, Anderson told ProteoMonitor, “because there are decades of experience using [it] to do really good quantitation on drugs, metabolites, hormones, everything.”

This history has also led Anderson to focus on SRM-MS in his SISCAPA research efforts, including in the assay development services being offered by his company SISCAPA Assay Technologies, which he launched in June (PM 6/17/2011). However, recent work being done independently by Stanford University proteomics researcher Mark Stolowitz and Morteza Razavi, a PhD candidate in the lab of University of Victoria professor and SISCAPA Assay Technologies co-founder Terry Pearson, suggest that MALDI-TOF mass spec might also prove a suitable platform.

MALDI-TOF machines are typically less expensive than high-end triple quads, and MALDI-MS assays are much easier and quicker to develop than SRM assays. Additionally, MALDI instruments can measure the amount of target peptide in a sample in a matter of seconds, compared to cycle times of minutes or tens of minutes for SRM assays. Such throughput is key to clinical applications where researchers need to process thousands of samples, either for validation studies or actual clinical use.

MALDI-TOF platforms have smaller dynamic ranges than triple quads, and typically don’t offer the high level of sensitivity and specificity that SRM assays provide, but, Anderson said, Razavi’s work suggests that in many cases SISCAPA’s antibody capture step “generates, effectively, a pure peptide analyte.”

“In that case, where the peptide sample we’re analyzing is pure, then you have to worry much less about the specificity of the mass spectrometer,” he said. “So, if we can generate antibodies that really give us a great purification of the target peptide, then we have a lot of flexibility in what mass spec platform we can use, and that opens up MALDI-TOF as a viable candidate.”

Thus far, Razavi said, he has generated MALDI-TOF-based SISCAPA assays for a variety of protein biomarkers linked to prostate cancer and has run them on around 250 clinical samples obtained from the BC Cancer Agency’s Deeley Research Centre.

His work on these assays has also addressed concerns that MALDI instruments suffer from high coefficients of variation when used for quantitation.

“The [proteomics] community, I feel, really doesn’t trust MALDI to have as good CVs as SRM mass spectrometry,” Razavi told ProteoMonitor. “But if you remain within the dynamic range of your analytes and collect enough counts for each target you are trying to measure, you can achieve great CVs [using MALDI] – in some cases even better than SRM-based mass spec.”

MALDI’s reputation for high variability stems from relative quantitation experiments people have done that produced CVs of 30 or 40 percent, Anderson said. “But it turned out that people had not really systematically investigated how good quantitation would be if you have stable isotope-labeled internal standards [as in SISCAPA],” he added.

In collaboration with Bruker, Anderson’s team investigated MALDI TOF-based quantitation using internal standards and found that they were able to achieve CVs below one percent in some cases. According to Razavi, the variability attributable to the mass spec analysis in the MALDI SISCAPA assays that he’s developed is around 3.5 percent. CVs for the entire workflow, including trypsin digestion, average under 15 percent, which he said is good enough for clinical use.

Sound Waves for Smaller Spots

One drawback that Razavi’s work has not been able to address is the low sensitivity of MALDI-TOF instruments compared to triple quads. His assays are able to measure proteins at concentrations of 100 nanograms per mL and higher, making them useful for quantifying medium- and high-abundance proteins but not low-abundance analytes.

Stanford’s Stolowitz, however, has developed a MALDI-based SISCAPA workflow using acoustic liquid handling technology from sample prep firm Labcyte that, he said, offers sensitivity on par with SRM assays on a triple quad.

Labcyte’s technology, which uses sound waves to create and manipulate minute liquid droplets, has great potential for use in creating MALDI matrices due to its ability to spot extremely small amounts of sample, Stolowitz, who is the director of the Proteomic Core Facility at Stanford’s Canary Center for Cancer Early Detection, told ProteoMonitor. This, he said, should reduce heterogeneity across MALDI matrix spots, reducing variability and improving the technology’s limits of quantitation.

“Presentation of the analyte is not uniform across [a given MALDI matrix spot],” Stolowitz said. “During the process of drying the MALDI matrix the analyte may become concentrated in the outer ring; it may become concentrated centrally; or it may become concentrated heterogeneously in what we call ‘hot’ spots and ‘cold’ spots.”

“But by making the spot very, very minute, it becomes very homogeneous with respect to its composition,” he said, noting that, while typical MALDI spots are several millimeters across, Labcyte’s liquid-handling systems can make MALDI spots as small as 150 microns in diameter.

In November, the National Cancer Institute awarded Labcyte $196,000 for its work with Stolowitz on the MALDI SISCAPA platform. Thus far, Stolowitz said, his team “can’t detect any significant difference in sensitivity” between MALDI-based SISCAPA assays run on a AB Sciex TOF/TOF 5800 and typical SRM-based assays.

“That would not be the case if we weren’t using these very small spots,” he said. “If we were using conventional spots we would possibly be an order of magnitude less sensitive than we are.”

Anderson and Stolowitz have been in contact about Stanford’s MALDI work, both said, and Anderson noted that the platform “looks very interesting.” For now, though, he said he has no plans to incorporate the Labcyte technology into the SISCAPA assay development work offered through SISCAPA Assay Technologies.

Anderson has been working with Agilent on an automated SRM-based SISCAPA platform (PM 2/11/2011), and has managed to bring down the assay’s cycle time from roughly 30 minutes to three minutes. Use of MALDI, he said, could bring that down to around 20 seconds. He noted, though, that Agilent’s RapidFire separations platform, which the company acquired when it purchased Biocius in March (PM 3/4/2011), could bring the cycle time for SISCAPA SRM runs down to the level of the MALDI-based assays.

SISCAPA Assay Technologies plans to continue its work with Agilent on the sample-prep automation and SRM-based SISCAPA assays, Anderson said, and is collaborating with Bruker on the MALDI version. The company plans to begin offering MALDI SISCAPA development services once a paper on the technology it has under review is accepted, he said.

Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.